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DNA Topoisomerase-Mediated Illegitimate Recombination
... Some work has also suggested that in Escherichia coli cleavage complexes can induce illegitimate recombination between two DNA segments (Ikeda, Moriya et al. 1981;Ikeda 1994;Ikeda, Shiraishi et al. 2004). One possible mechanism being the exchange of subunits between two cleavage complexes, followed by re-ligation. ...
... Gyrase was shown to be involved in the transfer of an antibiotic resistance cassette from a plasmid to lambda DNA in a packaging reaction, in the presence of a quinolone (Ikeda, Moriya et al. 1981;Ikeda, Aoki et al. 1982).This result was independent of any homologous recombination pathway in either the phage or bacterium (Ikeda, Moriya et al. 1981;Ikeda, Aoki et al. 1982). The authors suggested that gyrase-mediated recombination in one of two ways: either 1) by dissolution of the gyrase complex into heterodimers (GyrA:GyrB) that still had the DNA bound in the phosphotyrosyl bond, which could reassociate with another GyrA:GyrB heterodimer in a similar situation, allowing for recombination, or 2) by the formation of higher order oligomers (GyrA 4 GyrB 4 ) and interface swapping, which upon dissolution of the higher-order complex could result in subunit exchange and recombination (Ikeda 1994;Ikeda, Shiraishi et al. 2004). We suggest the former may be the cause of slower subunit exchange observed in the formation of GyrA and GyrA59 heterodimers over several days (Suppl. ...
DNA gyrase, a ubiquitous bacterial enzyme, is a type IIA topoisomerase formed by heterotetramerisation of 2 GyrA subunits and 2 GyrB subunits, to form the active complex. GyrA is usually found as a dimer in solution, whereas GyrB can exist as a monomer. DNA gyrase is able to loop DNA around the C-terminal domains (CTDs) of GyrA and pass one DNA duplex through a transient double-strand break (DSB) established in another duplex. This results in the conversion of a positive loop into a negative one, thereby introducing negative supercoiling into the bacterial genome, an activity essential for DNA replication and transcription. The strong protein interface in the GyrA dimer must be broken to allow passage of the transported DNA segment and it is generally assumed that the interface is usually stable and only opens when DNA is transported, to prevent the introduction of deleterious DSBs in the genome. In this paper we show that DNA gyrase can exchange its DNA-cleaving interfaces between two active heterotetramers. This so-called “subunit exchange” can occur within a few minutes in solution. We also show that bending of DNA by gyrase is essential for cleavage but not for DNA binding per se and favors subunit exchange. Subunit exchange is also favored by DNA wrapping and an excess of GyrB. We suggest that proximity, promoted by GyrB oligomerization and binding and wrapping along a length of DNA, between two heteroteramers favors rapid interface exchange. This exchange does not require ATP, can occur in the presence of fluoroquinolones, and raises the possibility of non-homologous recombination solely through gyrase activity. The ability of gyrase to undergo subunit exchange also explains how gyrase heterodimers, containing a single active-site tyrosine, can carry out double-strand passage reactions.
... The more recently discovered fluoroquinolone compounds, such as ciprofloxacin, possess an additional mode of killing that does not require ongoing protein synthesis (Howard et al., 1993a,b). Since gyrase can participate in quinolone-stimulated subunit exchange (Ikeda, 1994), it seemed possible that the second mode of ciprofloxacin action might involve direct dissociation of quinolonegyrase-DNA complexes. As a test for this idea, E. coli was treated with 1 mg/ml ciprofloxacin and supercoil constraint was examined in nucleoids. ...
... One is a chloramphenicol-insensitive mode of killing, which we postulate to involve dissociation of the gyrase subunits that constrain DNA ends in ciprofloxacin-gyrase-DNA complexes (Figure 10, step d). The idea of subunit dissociation emerged from Ikeda's observation that certain classes of illegitimate recombination can be explained by gyrase subunit exchange and that quinolone compounds stimulate this exchange (reviewed by Ikeda, 1994). Support for the gyrase dissociation idea is provided by the inability of chloramphenicol to block either the lethal action of ciprofloxacin (Figure 7) or the ciprofloxacin-depen-dent flattening of nucleoid ethidium titration curves ( Figure 6B). ...
DNA gyrase, the bacterial enzyme that supercoils DNA, is trapped on chromosomal DNA by the 4-quinolone compounds, as drug-gyrase complexes that contain DNA breaks. Examination of chromosomal DNA extracted from Escherichia coli indicated that bacteriostatic concentrations of oxolinic acid trap gyrase and block DNA synthesis without releasing broken DNA from gyrase-DNA complexes. Release, detected as free rotation of DNA in the presence of an intercalating dye, occurred only at high, bactericidal oxolinic acid concentrations. Release of DNA breaks and cell death were both blocked by chloramphenicol, an inhibitor of protein synthesis, suggesting that synthesis of additional protein activity is required to free the DNA ends. Ciprofloxacin, a more potent quinolone, released DNA breaks and killed cells even in the presence of chloramphenicol. It is proposed that this second, chloramphenicol-insensitive mode for release of DNA breaks and cell killing arises from dissociation of gyrase subunits. Ciprofloxacin also killed a gyrase (gyrA) mutant resistant to the prototype of quinolone, nalidixic acid, and created complexes on DNA detected by DNA fragmentation. This lethal effect of ciprofloxacin was eliminated by additional mutations mapping in parC, one of the two genes encoding topoisomerase IV. Thus, the fluoroquinolone compounds have two intracellular targets. In the absence of the gyrA mutation, the parC (CipR) allele did not by itself confer resistance to ciprofloxacin, indicating that gyrase is the major quinolone target in E. coli. These findings provide a molecular explanation for quinolone action in bacteria and a new way to study topoisomerase IV-chromosome interactions.
... This result was independent of any homologous recombination pathway in either the phage or bacterium (Ikeda et al., 1982;Ikeda et al., 1981). The authors suggested that gyrase-mediated recombination in one of two ways: either (1) by dissolution of the gyrase complex into heterodimers (GyrA:GyrB) that still had the DNA bound in the phosphotyrosyl bond, which could reassociate with another GyrA:GyrB heterodimer in a similar situation, allowing for recombination, or (2) by the formation of higher order oligomers (GyrA 4 GyrB 4 ) and interface swapping, which upon dissolution of the higher-order complex could result in subunit exchange and recombination (Ikeda, 1994;Ikeda et al., 2004). We suggest the former may be the cause of slower subunit exchange observed in the formation of GyrA and GyrA59 heterodimers over several days (Figure 2-figure supplement 2). ...
DNA gyrase, a ubiquitous bacterial enzyme, is a type IIA topoisomerase formed by heterotetramerisation of 2 GyrA subunits and 2 GyrB subunits, to form the active complex. DNA gyrase can loop DNA around the C-terminal domains (CTDs) of GyrA and pass one DNA duplex through a transient double-strand break (DSB) established in another duplex. This results in the conversion from a positive (+1) to a negative (–1) supercoil, thereby introducing negative supercoiling into the bacterial genome by steps of 2, an activity essential for DNA replication and transcription. The strong protein interface in the GyrA dimer must be broken to allow passage of the transported DNA segment and it is generally assumed that the interface is usually stable and only opens when DNA is transported, to prevent the introduction of deleterious DSBs in the genome. In this paper, we show that DNA gyrase can exchange its DNA-cleaving interfaces between two active heterotetramers. This so-called interface ‘swapping’ (IS) can occur within a few minutes in solution. We also show that bending of DNA by gyrase is essential for cleavage but not for DNA binding per se and favors IS. Interface swapping is also favored by DNA wrapping and an excess of GyrB. We suggest that proximity, promoted by GyrB oligomerization and binding and wrapping along a length of DNA, between two heterotetramers favors rapid interface swapping. This swapping does not require ATP, occurs in the presence of fluoroquinolones, and raises the possibility of non-homologous recombination solely through gyrase activity. The ability of gyrase to undergo interface swapping explains how gyrase heterodimers, containing a single active-site tyrosine, can carry out double-strand passage reactions and therefore suggests an alternative explanation to the recently proposed ‘swivelling’ mechanism for DNA gyrase (Gubaev et al., 2016).
... aureus [104]). A mechanism is suggested by Ikeda's attribution of quinolone-promoted illegitimate DNA recombination to gyrase subunit dissociation [105]: subunit dissociation could release the equivalent of double-strand breaks (Fig. (5e)) and thereby account for chloramphenicolinsensitive death. ...
Fluoroquinolone resistance among isolates of Streptococcus pneumoniae arises de novo in a stepwise manner through both target (DNA gyrase and DNA topoisomerase IV) and non-target mutations. Variants with intermediate susceptibility can increase the frequency at which highly resistant mutants are subsequently recovered; consequently, enrichment of moderately susceptible mutants is expected to accelerate the development of fluoroquinolone resistance. Consideration of the rate at which clinical susceptibility is being lost leads to a prediction of widespread fluoroquinolone resistance even though the absolute prevalence of resistance is currently low in many countries (about 1% for levofloxacin in the USA). In vitro experiments suggest that resistance might be restricted by dosing strategies that maintain fluoroquinolone concentrations high enough to prevent growth of mutant subpopulations. However, the animal and clinical tests required for implementation of those strategies have yet to be carried out. As the potential usefulness of the strategies for fluoroquinolone therapy with S. pneumoniae is likely to be eroded by existing treatment regimens, the time available for in vivo testing appears to be limited.
... The mechanisms of nonhomologous recombination are relatively poorly understood. However, a number of proteins such as topoisomerase I (Ikeda 1994), DNA gyrase (Waldman and Waldman 1990 ), poly(ADPribose )polymerase (Waldman and Waldman 1990), DNA ligase II (Waldman, and Waldman 1990), and nonhomologous recombination (NHR)ligase (Derbyshire et aL 1994), have been implicated to play very specific roles in illegitimate (nonhomologous) recombination. NHR ligase has the ability to join DNA duplexes, irrespective of the sequence and structure of their ends (Derbyshire et aL 1994). ...
Genetic recombination is the creation of new gene combinations in a cell or gamete, which differ from those of progenitor
cells or parental gametes. In eukaryotes, recombination may occur at mitosis or meiosis. Mitotic recombination plays an indispensable
role in DNA repair, which presumably directed its early evolution; the multiplicity of recombination genes and pathways may
be best understood in this context, although they have acquired important additional functions in generating diversity, both
somatically (increasing the immune repertoire) and in germ line (facilitating evolution). Chromosomal homologous recombination
and HsRad51 recombinase expression are increased in both immortal and preimmortal transformed cells, and may favor the occurrence of
multiple oncogenic mutations. Tumorigenesis in vivo is frequently associated with karyotypic instability, locus-specific gene rearrangements, and loss of heterozygosity at tumor
suppressor loci — all of which can be recombinationally mediated. Genetic defects which increase the rate of somatic mutation
(several of which feature elevated recombination) are associated with early incidence and high risk for a variety of cancers.
Moreover, carcinogenic agents appear to quite consistently stimulate homologous recombination. If cells with high recombination
arise, either spontaneously or in response to “recombinogens,” and predispose to the development of cancer, what selective
advantage could favor these cells prior to the occurrence of growth-promoting mutations? We propose that the augmentation of telomere-telomere recombination may
provide just such an advantage, to hyper-recombinant cells within a population of telomerase-negative cells nearing their
replicative (Hayflick) limit, by extending telomeres in some progeny cells and thus allowing their continued proliferation.
... That would allow GyrA-GyrB dimers, each attached to an end of cleaved DNA, to dissociate more readily. If that process is stimulated by quinolones, as appears to be the case when assayed by illegitimate recombination, 36 it would explain how some quinolones can be highly lethal even in the absence of protein synthesis. 37 Attempts to probe the GyrB-GyrB interaction have so far been inconclusive. ...
The B subunit of DNA gyrase (GyrB) consists of a 43 kDa N-terminal domain, containing the site of ATP binding and hydrolysis, and a 47 kDa C-terminal domain that is thought to play a role in interactions with GyrA and DNA. In cells containing a deletion of topA (the gene encoding DNA topoisomerase I) a compensatory mutation is found in gyrB. This mutation (gyrB-225) results in a two amino acid insertion in the N-terminal domain of GyrB. We found that cells containing this mutation are more sensitive than wild-type cells to quinolone drugs with respect to bacteriostatic and lethal action. We have characterised the mutant GyrB protein in vitro and found it to have reduced DNA supercoiling, relaxation, ATPase, and cleavage activities. The mutant enzyme is up to threefold more sensitive to quinolones than wild-type. The mutation also increases the affinity of GyrB for GyrA and DNA, while the affinity of quinolone for the enzyme-DNA complex is unaffected. We propose that the loss in activity is due to misfolding of the GyrB-225 protein, providing an example in which misfolding of one protein, DNA gyrase, suppresses a deficiency of another, topoisomerase I. The increased quinolone sensitivity is proposed to be a consequence of an altered conformation of the protein that renders quinolones better able to disrupt, rather than generate, gyrase-drug-DNA complexes.
... It has been appreciated for many years that gyrase can participate in IR reactions (113) and that this reaction is stimulated (2-3 orders of magnitude) by the quinolone drug oxolinic acid (114), which binds at the DNA gate. In contrast, this stimulation is blocked by the coumarin drug coumermycin A 1 , which prevents ATP binding (115). Gyrase-mediated IR can occur in vitro and requires the presence of an E. coli extract, but is independent of RecA (114). ...
Type II DNA topoisomerases (topos) catalyse changes in DNA topology by passing one double-stranded DNA segment through another.
This reaction is essential to processes such as replication and transcription, but carries with it the inherent danger of
permanent double-strand break (DSB) formation. All type II topos hydrolyse ATP during their reactions; however, only DNA gyrase
is able to harness the free energy of hydrolysis to drive DNA supercoiling, an energetically unfavourable process. A long-standing
puzzle has been to understand why the majority of type II enzymes consume ATP to support reactions that do not require a net
energy input. While certain type II topos are known to ‘simplify’ distributions of DNA topoisomers below thermodynamic equilibrium
levels, the energy required for this process is very low, suggesting that this behaviour is not the principal reason for ATP
hydrolysis. Instead, we propose that the energy of ATP hydrolysis is needed to control the separation of protein–protein interfaces
and prevent the accidental formation of potentially mutagenic or cytotoxic DSBs. This interpretation has parallels with the
actions of a variety of molecular machines that catalyse the conformational rearrangement of biological macromolecules.
... The experiments described above clearly demonstrated that inhibition of DNA topoisomerase II activity during DNA replication strongly stimulated the process of illegitimate DNA recombination. (44) At the same time, the ability of DNA topoisomerase II to mediate DNA transfer directly (i.e., via subunit exchange (45) ) was estimated to be very low. ...
Chromosomal rearrangements frequently occur at specific places ("hot spots") in the genome. These recombination hot spots are usually separated by 50-100 kb regions of DNA that are rarely involved in rearrangements. It is quite likely that there is a correlation between the above-mentioned distances and the average size of DNA loops fixed at the nuclear matrix. Recent studies have demonstrated that DNA loop anchorage regions can be fairly long and can harbor DNA recombination hot spots. We previously proposed that chromosomal DNA loops may constitute the basic units of genome organization in higher eukaryotes. In this review, we consider recombination between DNA loop anchorage regions as a possible source of genome evolution.
... Like SSA, NHEJ is a non-conservative repair pathway, as nucleotides adjacent to the DSB can be lost during the joining of the broken chromosome ends 15 . DSB repair involving two different chromosomes has the potential to promote genome rearrangements, including translocations, although the number of lesions needed to promote these events is controversial [3][4][5][6][7][8] . Also unknown is the role of various repair pathways in limiting these events in mammalian cells. ...
The faithful repair of DNA damage such as chromosomal double-strand breaks (DSBs) is crucial for genomic integrity. Aberrant repair of these lesions can result in chromosomal rearrangements, including translocations, which are associated with numerous tumours. Models predict that some translocations arise from DSB-induced recombination in differentiating lymphoid cell types or from aberrant repair of DNA damage induced by irradiation or other agents; however, a genetic system to study the aetiology of these events has been lacking. Here we use a mouse embryonic stem cell system to examine the role of DNA damage on the formation of translocations. We find that two DSBs, each on different chromosomes, are sufficient to promote frequent reciprocal translocations. The results are in striking contrast with interchromosomal repair of a single DSB in an analogous system in which translocations are not recovered. Thus, while interchromosomal DNA repair does not result in genome instability per se, the presence of two DSBs in a single cell can alter the spectrum of repair products that are recovered.
... aureus [104]). A mechanism is suggested by Ikeda's attribution of quinolone-promoted illegitimate DNA recombination to gyrase subunit dissociation [105]: subunit dissociation could release the equivalent of double-strand breaks (Fig. (5e)) and thereby account for chloramphenicolinsensitive death. ...
Fluoroquinolones trap gyrase and topoisomerase IV on DNA as ternary complexes that block the movement of replication forks and transcription complexes. Studies with resistant mutants indicate that during complex formation quinolones bind to a surface alpha-helix of the GyrA and ParC proteins. Lethal action is a distinct event that is proposed to arise from release of DNA breaks from the ternary complexes. Many bacterial pathogens are exhibiting resistance due to alterations in drug permeability, drug efflux, gyrase-protecting proteins, and target topoisomerases. When selection of resistant mutants is described in terms of fluoroquinolone concentration, a threshold (mutant prevention concentration, MPC) can be defined for restricting the development of resistance. MPC varies among fluoroquinolones and pathogens; when combined with pharmacokinetics, MPC can be used to identify compounds least likely to enrich mutant subpopulations. Use of suboptimal doses and compounds erodes the efficacy of the class as a whole because resistance to one quinolone reduces susceptibility to others and/or increases the frequency at which resistance develops. When using fluoroquinolones in combination therapy, the development of resistance may be minimized by optimizing regimens for pharmacokinetic overlap.
DNA gyrase, a ubiquitous bacterial enzyme, is a type IIA topoisomerase formed by heterotetramerisation of 2 GyrA subunits and 2 GyrB subunits, to form the active complex. GyrA is usually found as a dimer in solution, whereas GyrB can exist as a monomer. DNA gyrase is able to loop DNA around the C-terminal domains (CTDs) of GyrA and pass one DNA duplex through a transient double-strand break (DSB) established in another duplex. This results in the conversion of a positive loop into a negative one, thereby introducing negative supercoiling into the bacterial genome, an activity essential for DNA replication and transcription. The strong protein interface in the GyrA dimer must be broken to allow passage of the transported DNA segment and it is generally assumed that the interface is usually stable and only opens when DNA is transported, to prevent the introduction of deleterious DSBs in the genome. In this paper we show that DNA gyrase can exchange its DNA-cleaving interfaces between two active heterotetramers. This so-called “subunit exchange” can occur within a few minutes in solution. We also show that bending of DNA by gyrase is essential for cleavage but not for DNA binding per se and favors subunit exchange. Subunit exchange is also favored by DNA wrapping and an excess of GyrB. We suggest that proximity, promoted by GyrB oligomerization and binding and wrapping along a length of DNA, between two heteroteramers favors rapid interface exchange. This exchange does not require ATP, can occur in the presence of fluoroquinolones, and raises the possibility of non-homologous recombination solely through gyrase activity. The ability of gyrase to undergo subunit exchange also explains how gyrase heterodimers, containing a single active-site tyrosine, can carry out double-strand passage reactions.
This chapter describes topoisomerases. Much progress has been made in structural and functional studies on DNA topoisomerases. New enzymes of the topoisomerase family are still being discovered, and the biological implications of these enzymes stands under open discussion. The potential of DNA topoisomerases as drug targets has expanded this research field into the realm of pharmacology and clinical medicine. Some topics like the viral and mithocondrial topoisomerases remain largely unexplored. The chapter presents the current understanding on the structure and mechanism of these enzymes, and the biological roles of DNA topoisomerases in prokaryotic and eukaryotic cells. All known DNA topoisomerases share two characteristics. One is their ability to cleave and reseal the phosphodiester backbone of DNA in two successive transesterification reactions. The second characteristic is that once a topoisomerase-cleaved DNA intermediate is formed, the enzyme allows the severed DNA ends to come apart, opening a gate for the passage of another single- or double-stranded DNA segment.
Topoisomerase-targeting drugs are a vast group of compounds exerting various pharmacological activities, characterized by their ability to target the nuclear proteins topoisomerases or their prokaryotic analogues. This chapter briefly examine the topoisomerase I mechanism of action and its significance as a target for antitumor chemotherapy. The field of cancer pharmacology gained a new protagonist with camptothecin. Effort in understanding the molecular basis of its action, coupled to the interest in exploiting its antitumor properties, brought interaction between different disciplines in the basic sciences with the applied clinical sciences. The resulting effect has been synthetic development of derivatives with better pharmacokinetic profiles, and the evaluation of more efficient administration protocols. More derivatives are likely to be developed in the future: they will bring their contribution to the unveiling of the drugs mechanism of action or, if they find their way to the clinical practice, to the fight against cancer.
Abstract DNA gyrase is an essential bacterial enzyme that catalyzes the ATP-dependent negative super-coiling of double-stranded closed-circular DNA. Gyrase belongs to a class of enzymes known as topoisomerases that are involved in the control of topological transitions of DNA. The mechanism by which gyrase is able to influence the topological state of DNA molecules is of inherent interest from an enzymological standpoint. In addition, much attention has been focused on DNA gyrase as the intracellular target of a number of antibacterial agents and as a paradigm for other DNA topoisomerases. In this review we summarize the current knowledge concerning DNA gyrase by addressing a wide range of aspects of the study of this enzyme.
DNA ligation by DNA topoisomerase I was investigated employing synthetic DNA substrates containing a single strand nick. Site-specific cleavage of the DNA by topoisomerase I in proximity to the nick resulted in uncoupling of the cleavage and ligation reactions of the enzyme, thereby trapping the covalent enzyme−DNA intermediate. DNA cleavage could be reversed by the addition of acceptor oligonucleotides containing a free 5‘-OH group and capable of hybridizing to the noncleaved strand of the “suicide substrates”. Utilizing acceptors with partial complementarity, modification of nucleic acid structure has been obtained. Modifications included the formation of DNA insertions, deletions, and mismatches. To further evaluate the potential of topoisomerase I to mediate structural transformations of DNA, acceptor oligonucleotides containing nucleophiles other than OH groups at the 5‘-end were studied as substrates for the topoisomerase I-mediated ligation reaction. Toward this end, oligonucleotides containing 5‘-thio, amino, and hydroxymethylene moieties were synthesized. Initial investigations utilizing a coupled cleavage−ligation assay suggested that only the modified acceptor containing an additional methylene group underwent efficient enzyme-mediated ligation. However, as linear DNA is not a preferred substrate for topoisomerase I, the enzyme−DNA intermediate was purified to homogeneity, thereby allowing investigation of the ligation reaction independent of the forward reaction that formed the covalent binary complex. The isolated complex consisted of equimolar enzyme and DNA, with topoisomerase I covalently bound to a specific site on the DNA duplex in an enzymatically competent form. Displacement of the enzyme-linked tyrosine moiety of the enzyme−DNA binary complex was effected by all the modified acceptor oligonucleotides, affording unnatural internucleosidic linkages at a specific site. Characterization of the formed linkages was effected both by enzymatic and chemical degradation studies. Comparative analysis revealed overall differences in the efficiency and rate of the topoisomerase I-mediated ligation of the modified acceptors. Moreover, the facility of ligation of the amino acceptor was significantly enhanced at increasing pH values. In addition, the method utilized to obtain the topoisomerase I−DNA intermediate is capable of affording large quantities required for further mechanistic and physicochemical characterization of the formed binary complex.
As noted elsewhere in this book, the locus of action of camptothecin (CPT) (1) (Fig. 1) as an antitumor agent involves the noncovalent binding of this agent to the covalent binary complex formed between
topoisomerase I (TOP-I) and DNA (Fig. 2) (1). Although the equilibrium between free enzyme and the enzyme-DNA binary complex normally lies far toward free enzyme and
DNA, in the presence of CPT, the equilibrium is rapidly displaced toward ternary complex (2). At this level, the action of CPT on TOP-I function is entirely analogous to those of several agents that inhibit the function
of topoisomerase II, including 4-(9-acridinylamino)-N-(methanesulfonyl)-m-anisidine (m-AMSA), etoposide, and teniposide (3).
We have previously shown the presence, in the amplified DNA of a Drosophila cell line resistant to N-phosphonacetyl-L-aspartate (PALA), of two units of 150 kb and 120 kb respectively duplicated and amplified. The two joints (J1 and J2) linking these units as well as their respective wild-type counterparts have been sequenced. Sequence analysis indicates that a region of the Drosophila genome which corresponds to the proximal boundary of the 150 kb unit is common to both joints. In addition to this common region, the J1 junction possesses a 26-nucleotide sequence belonging to the J2 junction. This indicates that the J2 junction was the first formed, and that J1, therefore, results from recombination between J2 and a region of the wild-type genome 120 kb distal to J2. Sequence analysis also reveals that the joints result from illegitimate recombination between unrelated regions. AT-rich sequences, strand bias composition and putative topoisomerase I and II sites were found in at least one of the two parental sequences involved in the formation of the joints. On the basis of these results we can hypothesize that after two illegitimate recombinations between sister chromatids, leading first to J2 and then to J1, the amplification may have arisen by a series of homologous (unequal crossing-over) or illegitimate recombinations, or by an intrachromosomal rolling circle.
DNA topoisomerases have been proposed as the proteins involved in the formation of the DNA-protein cross-links detected after ultraviolet light (UV) irradiation of cellular DNA. This possibility has been investigated by studying the effects of UV-induced DNA damage on human DNA topoisomerase I action. UV lesions impaired the enzyme's ability to relax negatively supercoiled DNA. Decreased relaxation activity correlated with the stimulation of cleavable complexes. Accumulation of cleavable complexes resulted from blockage of the rejoining step of the cleavage-religation reaction. Mapping of cleavage sites on the pAT153 genome indicated UV-induced cleavage at discrete positions corresponding to sites stimulated also by the topoisomerase I inhibitor camptothecin, except for one. Subsequent analysis at nucleotide level within the sequence encompassing the UV-specific cleavage site revealed the precise positions of sites stimulated by camptothecin with respect to those specific for UV irradiation. Interestingly, one of the UV-stimulated cleavage sites was formed within a sequence that did not contain dimerized pyrimidines, suggesting transmission of the distortion, caused by photodamage to DNA, into the neighboring sequences. These results support the proposal that DNA structural alterations induced by UV lesions can be sufficient stimulus to induce cross-linking of topoisomerase I to cellular DNA.
Recently, it has been discovered that DNA topoisomerases are the target of many anti-cancer drugs which were already widely used in the clinic. Using the latest molecular biological techniques much has been learned about the function of DNA topoisomerases in normal cells and in cancer cells, and about how anticancer drugs inhibit these enzymes. In this review we present an overview of the function of human type II DNA topoisomerases, how they are inhibited by certain drugs and on how cancer cells may become resistant to these drugs.
Etoposide is one of the most widely used antineoplastics. Unfortunately, the same treatment schedules associated with impressive efficacy are associated with an increased risk of secondary acute myeloid leukemia (AML), which has prompted its withdrawal from some treatment regimens, thereby potentially compromising efficacy against the original tumor. Because etoposide-associated AML is characterized by site-specific illegitimate DNA recombination, we studied whether etoposide could directly cause site-specific deletions of exons 2 and 3 in the hprt gene. Human lymphoid CCRF-CEM cells were treated with etoposide for 4 hours, and DNA was isolated after subculturing. The deletion of exons 2 and 3 from hprt was assayed by a quantitative polymerase chain reaction (PCR) method. In the absence of etoposide treatment, the frequency of deletions of exons 2 and 3 was very low (5.05 x 10(-8)). After exposure to 10 mumol/ L etoposide, the frequency of the exon 2 + 3 deletion was increased immediately after and at 24 hours after etoposide treatment (65 to 89 x 10(-8)) and increased to higher levels (128 to 173 x 10(-8)) after 2 and 6 days of subculture (P < .001 overall). The frequency of the exon 2 + 3 deletion assessed at 6 days of subculture after 4 hours of 0, 0.25, 1, 2.5, 5, and 10 mumol/L etoposide treatment increased with etoposide concentration, ie, 5.05 x 10(-8), 89.2 x 10(-8), 108 x 10(-8), 142 x 10(-8), 163 x 10(-8), and 173 x 10(-8), respectively (P < .0001). Sequencing of a subset of amplified products confirmed the presence of DNA sequences at the breakpoints consistent with V(D)J recombination. By contrast, exon 2 + 3 deletions after etoposide treatment in the myeloid cell lines KG-1A and K562 showed no evidence of V(D)J recombinase in their genesis. We conclude that etoposide can induce the illegitimate site-specific action of V(D)J recombinase on an unnatural DNA substrate after a single treatment in human lymphoid cells.
To study the involvement of DNA topoisomerase (topo) II on nonhomologous (illegitimate) recombination, we examined the effect of topo II inhibitors on random integration of exogenous vectors into human chromosomes. We transfected human cell lines PA1, HeLa and EJ-1 with linearized plasmid pSV2neo by electroporation, treated with topo II inhibitors and determined the frequency of Geneticin-resistant (G418r) colonies. We found that three topo II inhibitors, etoposide (VP-16), ICRF-193 and amsacrine (m-AMSA), greatly enhanced the frequency of G418r colonies. These effects were maximally expressed by as little as 12 hrs treatment with the drugs. Similar enhancements were found with different vectors (closed-circular and linear), different cell types, or by different transfection methods (calcium precipitation and lipofection). In contrast, the inhibitor treatments did not affect the transient expression of chloramphenicol acetyltransferase and beta-galactosidase activity following transfection with pSV2CAT and pCH110, respectively. Southern blot analysis revealed that the integration pattern of transfected pSV2neo into PA1 chromosomes was random and not characteristic for each inhibitor. These results suggest that topo II inhibitors directly act at a nonhomologous recombination reaction, promoting the integration process of transfected vectors into human chromosomes. We discuss the enhancement mechanism with a special emphasis on DNA strand breaks induced by the inhibitors.
Formation of araB-lacZ coding-sequence fusions is a key adaptive mutation system. Eighty-four independent araB-lacZ fusions were sequenced. All fusions carried rearranged MuR linker sequences between the araB and lacZ domains indicating that they arose from the standard intermediate of the well-characterized Mu DNA rearrangement process, the strand transfer complex (STC). Five non-standard araB-lacZ fusions isolated after indirect sib selection had novel structures containing back-to-back inverted MuR linkers. The observation that different isolation procedures gave rise to standard and non-standard fusions indicates that cellular physiology can influence late steps in the multi-step biochemical sequence leading to araB-lacZ fusions. Each araB-lacZ fusion contained two novel of DNA junctions. The MuR-lacZ junctions showed 'hot-spotting' according to established rules for Mu target selection. The araB-MuR and MuR-MuR junctions all involved exchanges at regions of short sequence homology. More extensive homology between MuR and araB sequences indicates potential STC isomerization a resolvable four-way structure analogous to a Holliday junction. These results highlight the molecular complexity of araB-lacZ fusion formation, which may be thought of as a multi-step cell biology process rather than a unitary biochemical reaction.
A camptothecin-resistant cell line that exhibits more than 600-fold resistance to camptothecin, designated CPT(R)-2000, was established from mutagen-treated A2780 ovarian cancer cells. CPT(R)-2000 cells also exhibit 3-fold resistance to a DNA minor groove-binding ligand Ho33342, a different class of mammalian DNA topoisomerase I inhibitors. However, CPT(R)-2000 cells exhibit no cross-resistance toward drugs such as Adriamycin, amsacrine, vinblastine, and 4'-dimethyl-epipodophyllotoxin. The mRNA, protein levels, and enzyme-specific activity of DNA topoisomerase I are relatively the same in parental and CPT(R)-2000 cells. However, unlike the DNA topoisomerase I activity of parental cells, which can be inhibited by camptothecin, that of CPT(R)-2000 cells cannot. In addition, parental cells after camptothecin treatment results in a decrease in the level of DNA topoisomerase I, whereas CPT(R)-2000 cells are insensitive to camptothecin treatment. These results suggested that the mechanism of camptothecin resistance is most likely due to a DNA topoisomerase I structural mutation. This notion is supported by DNA sequencing results confirming that DNA topoisomerase I of CPT(R)-2000 is mutated at amino acid residues Gly717 to Val and Thr729 to Ile. We also used the yeast system to examine the mutation(s) responsible for camptothecin resistance. Our results show that each single amino acid change results in partial resistance, and the double mutation gives a synergetic effect on camptothecin resistance. Because both mutation sites are near the catalytic active center, this observation raises the possibility that camptothecin may act at the vicinity of the catalytic active site of the enzyme-camptothecin-DNA complex.
Type II DNA topoisomerases function as homodimeric enzymes in transiently cleaving double-stranded DNA to catalyze unlinking and unknotting reactions. The dimeric enzyme creates a DNA double-strand break by forming a covalent attachment between an active site tyrosine from each monomer and a 5'-phosphate from each strand of DNA. The dimer must be very stable to dissociation or subunit exchange when covalently attached to DNA to prevent directly or indirectly catalyzed rearrangements of the genome. Past studies have indicated conflicting results for the monomer-dimer stability of topoisomerase II in solution. Here, we report results from sedimentation equilibrium studies and two different subunit exchange assays indicating that purified Saccharomyces cerevisiae DNA topoisomerase II exists as a stable dimer in solution, with a Kd estimated to be < or = 10(-11) M. This high dimer stability is not detectably altered by a change of ionic strength or by the presence of ATP, ADP, or DNA.
Replication blockage induces non-homologous deletions in Escherichia coli. The mechanism of the formation of these deletions was investigated. A pBR322-mini-oriC hybrid plasmid carrying two E. coli replication terminators (Ter sites) in opposite orientations was used. Deletions which remove at least the pBR322 blocking site (named Ter1) occurred at a frequency of 2 x 10(-6) per generation. They fall into two equally large classes: deletions that join sequences with no homology, and others that join sequences of 3-10 bp of homology. Some 95% of the deletions in the former class resulted from the fusion of sequences immediately preceding the two Ter sites, indicating a direct role for blocked replication forks in their formation. These deletions were not found in a topA10 mutant, suggesting a topoisomerase I-mediated process. In contrast, deletions joining short homologous sequences were not affected by the topA10 mutation. However, the incidence of this second class of deletions increased 10-fold in a recD mutant, devoid of exonuclease V activity. This indicates that linear molecules are intermediates in their formation. In addition, approximately 50% of these deletions were clustered in the region flanking the Ter1 site. We propose that they are produced by repair of molecules broken at the blocked replication forks.
We have previously shown that the RAD50, RAD52, MRE11, XRS2, and HDF1 genes of Saccharomyces cervisiae are involved in the formation of deletions by illegitimate recombination on a monocentric plasmid. In this study, we investigated the effects of mutations of these genes on formation of deletions of a dicentric plasmid, in which DNA double-strand breaks are expected to occur frequently because the two centromeres are pulled to opposite poles in mitosis. We transformed yeast cells with a dicentric plasmid, and after incubation for a few division cycles, cells carrying deleted plasmids were detected using negative selection markers. Deletions occurred at a higher frequency than on the monocentric plasmid and there were short regions of homology at the recombination junctions as observed on the monocentric plasmid. In rad50, mre11, xrs2, and hdf1 mutants, the frequency of occurrence of deletions was reduced by about 50-fold, while in the rad52 mutant, it was comparable to that in the wild-type strain. The end-joining functions of Rad50, Mre11, Xrs2, and Hdf1, suggest that these proteins play important roles in the joining of DNA ends produced on the dicentric plasmid during mitosis.
Topotecan (Hycamtin) is a promising new topoisomerase I-targeting anticancer agent that first entered clinical trials in 1989 under National Cancer Institute sponsorship in collaboration with SmithKline Beecham. In 1996, it was approved for use by the United States Food and Drug Administration (FDA) for previously treated patients with advanced ovarian cancer. For these patients, topotecan provides another therapeutic option upon disease progression after initial platinum-based chemotherapy. Topotecan also has activity in other tumor types, including small-cell lung cancer, hematologic malignancies and pediatric neuroblastoma and rhabdomyosarcoma. Topotecan combination regimens with paclitaxel (Taxol), etoposide (VePesid), cisplatin (Platinol), and cytarabine and with other treatment modalities, such as radiation therapy, are in development. Studies evaluating topotecan combinations as initial treatment in such diseases as ovarian and small-cell lung carcinoma are also underway. It is hoped that earlier use of topotecan, with its novel mechanism of action, will prolong survival and increase cure rates in patients with these chemoresponsive tumors. Whether or not such hopes are realized, these important studies will help define the role of topotecan in cancer chemotherapy.
A partial DNA duplex containing a high efficiency topoisomerase I cleavage site was substituted singly at each of three sites with 3'-deoxyadenosine. Depending on the site of substitution, the facility of the topoisomerase I-mediated cleavage or ligation reactions was altered. Inclusion of the modified nucleoside at the 5'-end of the acceptor oligonucleotide diminished the rate of religation following substrate cleavage by the enzyme.
In mammalian cells, nonhomologous (illegitimate) recombination is a predominant pathway to repair DNA double-strand breaks. We have shown that DNA topoisomerase II inhibitors are capable of enhancing random integration of foreign DNA via nonhomologous recombination. Since this enhancement is likely due to stabilized DNA strand breaks, we examined the effect of a radiomimetic antitumor drug, bleomycin (BLM), on nonhomologous recombination. We found that BLM greatly enhances the random integration of transfected plasmids into human cells. Importantly, this enhancement was independent of the molecular form of the plasmid, the cell type or the transfection method, suggesting that the BLM effect is intrinsically general. Transient expression analysis revealed no stimulation of reporter gene expression by the drug, suggesting that the effect is not attributable to increased uptake and/or accumulation of transfected DNA in the drug-treated cell nuclei. In addition, the comet assay and flow cytometric analyses revealed the occurrence of low but significant strand breaks in cells treated with the BLM concentration which maximally enhanced the integration. These results strongly suggest that BLM acts directly at a nonhomologous recombination reaction that is initiated through DNA strand breaks, promoting the integration process of transfected plasmids into human chromosomes. Our findings will facilitate the understanding of DNA integration events through nonhomologous recombination and the development of transfection protocols with higher efficiencies.
Camptothecin (CPT) and 10 structural analogues were studied to characterize their effects on specific rearrangements of DNA structure mediated by human and calf thymus DNA topoisomerases I. A 30 base pair DNA duplex containing a single high-efficiency topoisomerase cleavage site was incubated with each of the enzymes in the presence of the inhibitors. Individual inhibitors stabilized the covalent enzyme-DNA binary complex to different extents, as anticipated. However, for several of the inhibitors, the extent of ternary complex formation differed substantially for the human and calf thymus enzymes. In common with calf thymus topoisomerase I, the human enzyme was shown to mediate the rearrangement of branched, nicked, and gapped DNA substrates that constitute models for illegitimate recombination. However, some of these rearrangements proceeded with different rates and efficiencies in the presence of human topoisomerase I. When inhibition of three of the rearrangements by CPT analogues was studied, most of the analogues exhibited differential effects on a given transformation, depending on the source of the enzyme employed.
DNA topoisomerases are double-edged swords. They are essential for many vital functions of DNA during normal cell growth. However, they are also highly vulnerable under various physiological and nonphysiological stresses because of their delicate act on breaking and rejoining DNA. These stresses (e.g. exposure to topoisomerase poisons, acidic pH, and oxidative stresses) can convert DNA topoisomerases into DNA-breaking nucleases, resulting in cell death and/or genomic instability. The importance of topoisomerase-mediated DNA cleavage in tumor cell death and carcinogenesis has been recognized. This review focuses on recent findings concerning the molecular mechanisms of the stress responses to topoisomerase-mediated DNA damage. The involvement of ubiquitin/26S proteasome and SUMO/UBC9 in these processes, as well as the role of topoisomerase cleavable complexes in apoptotic cell death are discussed.
Transfected linear DNA molecules are substrates for double-strand break (DSB) repair in mammalian cells. The DSB repair process can involve recombination between the transfected DNA molecules, between the transfected molecules and chromosomal DNA, or both. In order to determine whether these different types of repair events are linked, we devised assays enabling us to follow the fate of linear extrachromosomal DNA molecules involved in both interplasmid and chromosome-plasmid recombination, in the presence or absence of a pre-defined chromosomal DSB. Plasmid-based vectors were designed that could either recombine via interplasmid recombination or chromosome-plasmid recombination to produce a functional beta-galactosidase (betagal) fusion gene. By measuring the frequency of betagal+ cells at 36 h post-transfection versus the frequency of betagal+ clones after 14 days, we found that the number of cells containing extrachromosomal recombinant DNA molecules at 36 h (i.e., betagal+), either through interplasmid or chromosome-plasmid recombination, was nearly the same as the number of cells integrating these recombinant molecules. Furthermore, when a predefined DSB was created at a chromosomal site, the extrachromosomal recombinant DNA molecules were shown to integrate preferentially at that site by Southern and fiber-FISH (fluorescence in situ hybridization) analysis. Together these data indicate that the initial recombination event can potentiate or commit extrachromosomal DNA to integration in the genome at the site of a chromosomal DSB. The efficiency at which extrachromosomal recombinant molecules are used as substrates in chromosomal DSB repair suggests extrachromosomal DSB repair can be coupled to the repair of chromosomal DSBs in mammalian cells.
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Genetic integrity relies on the faithful repair of DNAdamage such as double-strand breaks (DSBs). Aberrantrepair of these lesions is expected to result in chromosomal rearrangements, including translocations, that are associated with numerous tumors (Rabbitts 1994; Mitelmanet al. 1997). Models predict that some translocations arisefrom DSB-induced recombination in differentiating lymphoid cell types (Lewis 1994; Hiom et al. 1998) or fromaberrant repair of DNA damage induced by irradiation orother agents (Cornforth and Bedford 1993; Ikeda 1995;Wang et al. 1997). Recently, a genetic system has beendeveloped to study the etiology of these events in mammalian cells (Richardson and Jasin 2000). This system relies on the expression of the rare-cutting I-SceI endonuclease (Jasin 1996) and the strategic placement of I-SceIcleavage sites in the genome to introduce DSBs at predetermined genomic locations. With this system, it has beenshown that two DSBs are sufficient to cause reciprocalchromosomal translocations, even in repair-proficientcells (Richardson and Jasin 2000)...
This review provides a detailed discussion of recent advances in the medicinal chemistry of camptothecin, a potent antitumor antibiotic. Two camptothecin analogues are presently approved for use in the clinic as antitumor agents and several others are in clinical trials. Camptothecin possesses a novel mechanism of action involving the inhibition of DNA relaxation by DNA topoisomerase I, and more specifically the stabilization of a covalent binary complex formed between topoisomerase I and DNA. This review summarizes the current status of studies of the mechanism of action of camptothecin, including topoisomerase I inhibition and additional cellular responses. Modern synthetic approaches to camptothecin and several of the semi-synthetic methods are also discussed. Finally, a systematic evaluation of novel and important analogues of camptothecin and their contribution to the current structure-activity profile are considered.
Chromosomal rearrangements are common causes of cancer. In the majority of cases, the malignancy is induced via an altered transcription factor. The breakpoints of such translocations are often mysteriously tightly clustered in the genome. Even more surprisingly, such breakpoint clusters often contain specific genomic elements, such as topoisomerase II consensus sites, nuclear matrix attachment regions, etc. In this review, we discuss the common idea of breakpoints being induced by chromatin structure. We also touch on the question of whether the structure of corresponding proteins is related to the positions of breakpoints. Finally, we refer to recent works on chromosome territories and their distribution in the interphase nucleus.
Current biomedical research has its focus on the search for newer intervention strategies to control public health impact of parasitic diseases. The dramatic advances of molecular and cellular biology in recent times have provided opportunities for discovering and evaluating molecular targets for drug designing, which now form a rational basis for the development of improved anti parasitic therapy. DNA topoisomerases, the "cellular magicians" involved in nearly all biological processes governing DNA, have emerged as one such biological target. Over the last two decades, interest in topoisomerases has expanded beyond the realm of the basic science laboratory into the clinical arena. This review aims at providing a comprehensive insight into the biology of DNA topoisomerases and also focus on its evolution as a drug target in the unicellular kinetoplastids.
We have developed a plasmid test system to study recombination in vitro and in mammalian cells in vivo, and to analyze the possible role of DNA topoisomerase II. The system is based on a plasmid construct containing an inducible marker gene ccdB ("killer" (KIL) gene) whose product is lethal for bacterial cells, flanked by two different potentially recombinogenic elements. The plasmids were subjected to recombinogenic conditions in vitro or in vivo after transient transfection into COS-1 cells, and subsequently transformed into E. coli which was then grown in the presence of the ccdB gene inducer. Hence, all viable colonies contained recombinant plasmids since only recombination between the flanking regions could remove the KIL gene. Thus, it was possible to detect recombination events and to estimate their frequency. We found that the frequency of topoisomerase II-mediated recombination in vivo is significantly higher than in a minimal in vitro system. The presence of VM-26, an inhibitor of the religation step of the topoisomerase II reaction, increased the recombination frequency by 60%. We propose that cleavable complexes of topoisomerase II are either not religated, triggering error-prone repair of the DNA breaks, or are incorrectly religated resulting in strand exchange. We also studied the influence of sequences known to contain preferential breakpoints for recombination in vivo after chemotherapy with topoisomerase II-targeting drugs, but no preferential stimulation of recombination by these sequences was detected in this non-chromosomal context.
We report here the large scale purification of DNA topoisomerase II from calf thymus glands, using the unknotting of naturally knotted P4 phage DNA as an assay for enzymatic activity. Topoisomerase II was purified more than 1300-fold as compared to the whole cell homogenate, with 22% yield. Analysis of the purified enzyme by sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed two bands of apparent molecular masses of 125 and 140 kDa. Tryptic maps of the two bands indicated that they derive from the same protein. Using these fragments, specific polyclonal antisera to topoisomerase II were raised in rabbits. Immunoblotting of whole cell lysates from various species indicated that topoisomerase II is well conserved among mammals and has a native subunit molecular mass of 180 kDa. Analytical sedimentation and gel filtration were used to determine a sedimentation coefficient of 9.8 S and a Stokes radius of 68 A. The calculated solution molecular mass of 277 kDa implies a dimer structure in solution. The purified topoisomerase II unknots P4 DNA in an ATP-dependent manner and is highly stimulated in its relaxation activity by ATP. A DNA-stimulated ATPase activity, as has been found with other type II topoisomerases, is associated with the purified enzyme. Approximate kinetic parameters for the ATPase reaction were determined to be: a Vmax of 0.06 nmol of ATP/(micrograms of protein) (min) and Km of 0.2 mM in the absence of DNA, and a Vmax of 0.2 nmol of ATP/(micrograms of protein) (min) and Km of 0.4 mM ATP in the presence of supercoiled plasmid DNA.
Escherichia coli DNA gyrase catalyzes negative supercoiling of closed duplex DNA at the expense of ATP. Two additional activities of the enzyme
that have illuminated the energy coupling component of the supercoiling reaction are the DNA-dependent hydrolysis of ATP to
ADP and Pi and the alteration by ATP of the DNA site specificity of the gyrase cleavage reaction. This cleavage of both DNA strands
results from treatment with sodium dodecyl sulfate of the stable gyrase-DNA complex that is trapped by the inhibitor oxolinic
acid. Either ATP or a nonhydrolyzable analogue, adenyl-5′-yl-imidodiphosphate (App[NH]p), shifts the primary cleavage site
on ColE1 DNA. The prevention by novobiocin and coumermycin A1 of this cleavage rearrangement places the site of action of the antibiotics at a reaction step prior to ATP hydrolysis. The
step blocked is the binding of ATP because coumermycin A1 and novobiocin interact competitively with ATP in the ATPase and supercoiling assays; the Ki values are more than four orders of magnitude less than the Km for ATP. This simple mechanism accounts for all effects of the drugs on DNA gyrase. Studies with App[NH]p, another potent
competitive inhibitor of reactions catalyzed by gyrase, show that cleavage of a high energy bond is not required for driving
DNA into the higher energy supercoiled form. With substrate levels of gyrase, App[NH]p induces supercoiling that is proportional
to the amount of enzyme; a -0.3 superhelical turn was introduced per gyrase protomer A. We postulate that ATP and App[NH]p
are allosteric effectors of a conformational change of gyrase that leads to one round of supercoiling. Nucleotide dissociation
favored by hydrolysis of ATP returns gyrase to its original conformation and thereby permits enzyme turnover. Such cyclic
conformational changes accompanying alteration in nucleotide affinity also seem to be a common feature of energy transduction
in other diverse processes including muscle contraction, protein synthesis, and oxidative phosphorylation.
Illegitimate recombination dependent on DNA gyrase in a cell-free system has previously been described. We have now mapped DNA gyrase cleavage sites in the vicinity of known recombination sites in pBR322. Among five recombination sites examined, three were found to coincide with a DNA gyrase cleavage site. This result suggests that the cleavage of DNA by DNA gyrase has a central role in the recombination process.
Integrative recombination of bacteriophage lambda requires the action of the protein Int, the product of the phage int gene. In this paper we show that highly purified Int relaxes supercoiled DNA. The association of this nicking-closing activity with Int is shown by: (i) the cosedimentation of nicking-closing and recombination activities of purified Int, (ii) the parallel inactivation of the two activities in purified Int by both heat and a specific antiserum, and (iii) the alteration of both activities in crude extracts of a strain expressing a mutant int gene. The nicking-closing activity of Int functions in the absence of divalent cations and in the absence of an apparent source of chemical energy. The activity displays no obvious sequence specificity and is inhibited by Mg2+, spermidine, and single-stranded DNA. Int relaxes positive as well as negative supercoils. We present a model for the mechanism of strand exchange that describes how the nicking-closing activity of Int might be used during recombination.
Extensively purified DNA gyrase from Escherichia coli is inhibited by nalidixic acid and by novobiocin. The enzyme is composed of two subunits, A and B, which were purified as separate components. Subunit A is the product of the gene controlling sensitivity to nalidixic acid (nalA) because: (i) the electrophoretic mobility of subunit A in the presence of sodium dodecyl sulfate is identical to that of the 105,000-dalton nalA gene product; (ii) mutants that are resistant to nalidixic acid (nalA(r)) produce a drug-resistant subunit A; and (iii) wild-type subunit A confers drug sensitivity to in vitro synthesis of varphiX174 DNA directed by nalA(r) mutants. Subunit B contains a 95,000-dalton polypeptide and is controlled by the gene specifying sensitivity to novobiocin (cou) because cou(r) mutants produce a novobiocin-resistant subunit B and novobiocin-resitant gyrase is made drug sensitive by wild-type subunit B. Subunits A and B associate, so that gyrase was also purified as a complex containing 105,000- and 95,000-dalton polypeptides. This enzyme and gyrase reconstructed from subunits have the same drug sensitivity, K(m) for ATP, and catalytic properties. The same ratio of subunits gives efficient reconstitution of the reactions intrinsic to DNA gyrase, including catalysis of supercoiling of closed duplex DNA, relaxation of supercoiled DNA in the absence of ATP, and site-specific cleavage of DNA induced by sodium dodecyl sulfate.
Escherichia coli DNA gyrase catalyzes negative supercoiling of closed duplex DNA at the expense of ATP. Two additional activities of the enzyme that have illuminated the energy coupling component of the supercoiling reaction are the DNA-dependent hydrolysis of ATP to ADP and P(i) and the alteration by ATP of the DNA site specificity of the gyrase cleavage reaction. This cleavage of both DNA strands results from treatment with sodium dodecyl sulfate of the stable gyrase-DNA complex that is trapped by the inhibitor oxolinic acid. Either ATP or a nonhydrolyzable analogue, adenyl-5'-yl-imidodiphosphate (App[NH]p), shifts the primary cleavage site on ColE1 DNA. The prevention by novobiocin and coumermycin A(1) of this cleavage rearrangement places the site of action of the antibiotics at a reaction step prior to ATP hydrolysis. The step blocked is the binding of ATP because coumermycin A(1) and novobiocin interact competitively with ATP in the ATPase and supercoiling assays; the K(i) values are more than four orders of magnitude less than the K(m) for ATP. This simple mechanism accounts for all effects of the drugs on DNA gyrase. Studies with App[NH]p, another potent competitive inhibitor of reactions catalyzed by gyrase, show that cleavage of a high energy bond is not required for driving DNA into the higher energy supercoiled form. With substrate levels of gyrase, App[NH]p induces supercoiling that is proportional to the amount of enzyme; a -0.3 superhelical turn was introduced per gyrase protomer A. We postulate that ATP and App[NH]p are allosteric effectors of a conformational change of gyrase that leads to one round of supercoiling. Nucleotide dissociation favored by hydrolysis of ATP returns gyrase to its original conformation and thereby permits enzyme turnover. Such cyclic conformational changes accompanying alteration in nucleotide affinity also seem to be a common feature of energy transduction in other diverse processes including muscle contraction, protein synthesis, and oxidative phosphorylation.
We have shown previously a good correlation between etoposide-induced sister chromatid exchanges (SCE) and cytotoxicity. A semisynthetic derivative of podophyllotoxin, etoposide is also called Vepesid (Bristol; code designation VP-16-213, abbreviated VP-16). Since SCE represent DNA recombinational events, we hypothesized that VP-16-induced SCE might result in nonhomologous recombination in which segments of DNA were either deleted or added, leading to an alteration of gene sequences responsible for essential cell proteins. Alterations of such essential genes and consequent interference with formation of their products could consequently lead to cell death. To evaluate whether VP-16 treatment caused sufficient levels of DNA sequence alterations to interfere with gene product formation, we isolated hypoxanthine (guanine) phosphoribosyltransferase (HPRT)-deficient mutants from Chinese hamster V79 cells grown in the presence or absence of VP-16. DNA from 3 spontaneous mutants and 10 VP-16-induced mutants was analyzed by Southern blot hybridization to a full-length hamster HPRT cDNA probe. Most of the VP-16-induced mutants showed partial deletions and/or rearrangements of the HPRT gene. In contrast, spontaneous mutants showed negligible deletions or rearrangements. These results provide strong support for our hypothesis that deletion of genetic sequences may constitute an important component of the mechanism of VP-16-induced cell death.
Cleavage of linear duplex DNA by purified vaccinia virus DNA topoisomerase I occurs at a conserved sequence element (5'-C/T)CCTT decreases) in the incised DNA strand. Oligonucleotides spanning the high affinity cleavage site CCCTT at nucleotide 2457 in pUC19 DNA are cleaved efficiently in vitro, but only when hybridized to a complementary DNA molecule. As few as 6 nucleotides proximal to the cleavage site and 6 nucleotides downstream of the site are sufficient to support exclusive cleavage at the high affinity site (position +1). Single nucleotide substitutions within the consensus pentamer have deleterious effects on the equilibria of the topoisomerase binding and DNA cleavage reactions. The effects of base mismatch within the pentamer are more dramatic than are the effects of mutations that preserve base complementarity. Competition experiments indicate that topoisomerase binds preferentially to DNA sites containing the wild-type pentamer element. Single-stranded DNA containing the sequence CCCTT in the cleaved stand is a more effective competitor than is single-stranded DNA containing the complementary sequence in the noncleaved strand.
Using site-specific mutagenesis in vitro we constructed a genetic system to detect mutants with altered rates of deletion formation between short repeated sequences in Escherichia coli. After in vivo mutagenesis with chemical mutagens and transposons, the system allowed the identification of mutants with either increased or decreased deletion frequencies. One mutational locus, termed mutR, that results in an increase in deletion formation, was studied in detail. The mutR gene maps at 38.5 min on the E. coli genetic map. Since the precise excision of many transposable elements is also mediated at short repeated sequences, we investigated the effects of the mutant alleles, as well as recA, on precise excision of the transposon Tn9. Neither mutR nor recA affect precise excision of the transposon Tn9, from three different insertions in lacI, whereas these alleles do affect other spontaneous deletions in the same system. These results indicate that deletion events leading to precise excision occur principally via a different pathway than other random spontaneous deletions. It is suggested that, whereas precise excision occurs predominantly via a pathway involving replication enzymes (for instance template strand slippage), deletions on an F'factor are stimulated by recombination enzymes.
A strong mutator effect has been observed in Escherichia coli K-12 strains mutated in the bglY gene (27 min). The frequency of point mutations is not modified in bglY mutant strains. In contrast, a strong increase in spontaneous generation of large deletions has been observed in these strains, both for chromosomal markers (10-fold increase of galETK-chlA deletions and 100-fold increase of ptsI-cysK deletions) and for plasmid DNA (100-fold increase of large deletions in the region located upstream of the chloramphenicol-resistance gene in plasmid pGR71). bglY mutations are recessive and can be complemented by a DNA fragment of 900 base pairs assumed to contain the entire bglY wild-type gene. This mutator effect is recA-independent.
We have previously shown that purified T4 DNA topoisomerase promotes illegitimate recombination between two lambda DNA molecules, or between lambda and plasmid DNA in vitro (Ikeda, H. (1986) Proc. Natl. Acad. Sci. U. S. A. 83, 922-926). Since the recombinant DNA contains a duplication or deletion, it is inferred that the cross-overs take place between nonhomologous sequences of lambda DNA. In this paper, we have examined the sequences of the recombination junctions produced by the recombination between two lambda DNA molecules mediated by T4 DNA topoisomerase. We have shown that there is either no homology or there are 1-5-base pair homologies between the parental DNAs in seven combinations of lambda recombination sites, indicating that homology is not essential for the recombination. Next, we have shown an association of the recombination sites with the topoisomerase cleavage sites, indicating that a capacity of the topoisomerase to make a transient double-stranded break in DNA plays a role in the illegitimate recombination. A consensus sequence for T4 topoisomerase cleavage sites, RNAY decreases NNNNRTNY, was deduced. The cleavage experiment showed that T4 topoisomerase-mediated cleavage takes place in a 4-base pair staggered fashion and produces 5'-protruding ends.
This chapter presents a historical account of the known episomes other than phage as well as some instances of possibly similar elements in higher organisms. Most of the episomes of bacteria fall into one of two classes—namely, temperate bacteriophages and transfer factors that can pass from cell to cell during conjugation independently of the bulk of the bacterial genome. Some transfer factors play a causative role in the conjugation process itself, such as the fertility factor of Escherichia coli K12. The episomic nature of temperate bacteriophage was established earlier than that of the transfer factors. A large amount of chemical and genetic work has converged to form a picture of intracellular bacteriophage growth, which is satisfying at a certain level. Much of the work on episomes and gene pick-up depends on the formation of partially diploid strains. The discovery of such strains antedates the findings with episomes, and there are some cases in which no known episome is implicated. Episomes consist of genetic material and frequently affect the intercellular transfer of other genetic material. The transfer of one episome by another reflects a physical connection of the two, so that at least during the time of transfer one episome behaves as part of the other.
Specialized transducing phages tna (tryptophanase) harboring chromosomal DNA and genetic markers from the dnaA region of the Escherichia coli chromosome were isolated. Transductional analysis showed that some of these tnaA transducing phages carry two genes important in DNA replication, namely the dnaA gene (initiation of chromosome replication) and the gyrB gene (subunit B of DNA gyrase), formerly designated cou
R. The following clockwise order of genetic markers was found:
uhp, gyrB, dnaA, rimA, tnaA, bglB.The gene-protein relationship was established by the determination of the gene products encoded on the chromosomal DNA of the different tna. A 54 kD and a 91 kD polypeptide appear to be coded for by the dnaA and gyrB genes, respectively; the 91 kD protein is encoded on a region in which coumermycin sensitivity maps and is with respect to electrophoretic behavior identical to subunit B of DNA gyrase. The 54 kD protein is encoded on the region in which different independently isolated dnaA(Ts) mutations (dnaA5, dnaA46, dnaA167, dnaA203, dnaA204, dnaA205, dnaA211, dnaA508) are located. Additional genes which code for polypeptides with hitherto unknown functions were identified and mapped. The acriflavin sensitivity mutation acrB1 was found to be an allele of the gyrB gene (see Note Added in Proof).
Illegitimate recombination dependent on T4 DNA topoisomerase in a cell-free system has recently been described. In that work, recombinants between two phage DNA molecules were produced by the topoisomerase alone, without an Escherichia coli extract. In this paper, it is shown that recombination between phage and circular plasmid DNA molecules can also be detected in the presence or absence of an E. coli extract but at frequencies two or three orders of magnitude lower than that observed in the phage-phage cross. The frequency is probably lower because multiple recombination is required in the case of the phage-plasmid cross.
To examine the mechanism of recombination involved in the formation of specialized transducing phage during the induction of bacteriophage we have determined the nucleotide sequences of the recombination junctions of bio phages. The results indicate that abnormal excision takes place at many sites on both bacterial and phage genomes and that the recombination sites have short regions of homology (5–14 bp). Some of the sequences of the recombination sites were similar to the consensus sequences of DNA gyrase-cleavage sites and repetitive extragenic palindromic (REP) sequences. These results showed that abnormal excision is a type of illegitimate recombination. The possible involvement of DNA gyrase in this recombination is discussed.
DNA gyrase has been purified to near homogeneity from Escherichia coli. The enzyme consists of two subunits of molecular weights 90,000 and 100,000 present in roughly equimolar amounts. The subunits can be identified as the products of two genes, determining resistance to coumermycin A1 and novobiocin (cou) and to nalidixic acid and oxolinic acid (nalA), respectively. These antibiotics were previously shown to be specific inhibitors of DNA gyrase. The ATPase activity of DNA gyrase is stimulated by double-stranded DNA and strongly inhibited by novobiocin but is relatively insensitive to oxolinic acid. Covalent attachment of an ATP derivative to the smaller (coumermycin-specific) subunit is also inhibited by novobiocin, suggesting that this drug interferes with the energy-coupling aspect of the DNA supercoiling reaction by blocking the access of ATP to the enzyme.
Relaxed closed-circular DNA is converted to negatively supercoiled DNA by DNA gyrase. This enzyme has been purified from Escherichia coli cells. The reaction requires ATP and Mg++ and is stimulated by spermidine. The enzyme acts equally well on relaxed closed-circular colicin E1, phage lambda, and simian virus 40 DNA. The final superhelix density of the DNA can be considerably greater than that found in intracellularly supercoiled DNA.
A target protein for nalidixic and oxolinic acids in Escherichia coli, the nalA gene product (Pnal), was purified to homogeneity as judged by gel electrophoresis, using an in vitro complementation assay. It is a dimer of identical 110,000-dalton subunits. A polypeptide of this molecular weight is uniquely induced by a lambda nalA transducing phage, thereby showing that the purified Pnal is a product of the nalA gene. Nalidixic and oxolinic acids inhibit DNA gyrase activity and induce formation of a relaxation complex analogue. Treatment of the complex with sodium dodecyl sulfate causes a doublestrand break in the DNA substrate and the resulting linear molecule seems covalently bound to protein. Complex formation, unlike the introduction of supertwists, does not require ATP or relaxed circular DNA and is insensitive to novobiocin. DNA gyrase from a strain with a nalA mutation conferring drug resistance (nalA(r)) is 1/100 as sensitive to oxolinic and nalidixic acids with respect to inhibition of supertwisting and induction of the pre-linearization complex. Addition of Pnal restores drug sensitivity and stimulates DNA gyrase activity. DNA gyrase preparations and Pnal catalyze a third reaction sensitive to nalidixic and oxolinic acids, the ATP-independent relaxation of supertwister DNA. Relaxation by gyrase from nalA(r) cells is drug resistant. The nicking-closing activity is distinct from E. coli omega protein in several properties, including the ability to relax positively supertwisted DNA. We postulate that the nalA gene product occurs in two molecular forms, as Pnal and as a gyrase component. Both forms catalyze nicking-closing, and inhibition of this activity by nalidixic and oxolinic acids may account for the inhibition of DNA synthesis by these drugs.
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Bacteriophage f1 is a filamentous phage that infects male strains of Escherichia coli. It is closely related to phages M13 and fd. All three phages contain a covalently circular, single-stranded (SS) DNA of 2 × 10⁶ daltons as their genome. Genetic analyses have demonstrated the existence of eight genes (Pratt et al. 1969), which have been ordered on a circular map (Lyons and Zinder 1972). Only two phage genes are involved in DNA replication: gene II, which is required for the replication of both replicative form (RF) DNA and viral SS DNA, and gene V, which codes for an SS DNA-binding protein that is required for the synthesis of viral SS DNA.
Phenomenologically, replication of f1 DNA occurs in three steps (for review, see Denhardt 1975; Ray 1977): (1) Conversion of the parental viral DNA (+ strand) into double-stranded (DS) RF DNA (parental RF DNA) by the synthesis of the...
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Double-stranded circular DNA, when isolated from cells or virions, is generally found to be supercoiled. The direction of supercoiling is always the same; the DNA strands are wound around each other too few times to permit a relaxed conformation to exist. This DNA structure is termed underwound, or negatively supercoiled. Until recently it has not been possible to determine how this supercoiling is generated. Two general classes of models, which may be termed passive and active, have been discussed.
In the passive model, intracellular DNA is distorted but relaxed, as judged by the criterion that nicking and resealing of a phosphodiester bond leads to no change in conformation. The distortion may be produced by the binding of proteins or other molecules. Purification of the DNA removes the bound molecules and allows the DNA to return to its free conformation, with the result that the distortions of helical winding now have...
Both the introduction and the removal of supertwists by DNA gyrase change the linking number of DNA in steps of two. This surprising finding provides strong evidence that gyrase acts by a mechanism, called sign inversion, whereby a positive supercoil is directly inverted into a negative one via a transient double-strand break.
Under some conditions, T4 DNA replication requires the products of the DNA-delay genes, genes 39, 52, 58, and 60. By using an in vitro complementation assay that stimulates DNA replication in T4 39(-)-infected cell extracts, T4 gene 39 protein has been purified. The purified fraction also contains complementing activities for T4 genes 52 and 60. On sodium dodecyl sulfate/polyacrylamide gel analysis the purified preparation exhibits three protein components: a 51,000-dalton protein corresponding to the product of gene 52, a 64,000-dalton protein corresponding to the product of gene 39, and a 110,000-dalton protein. This purified fraction shows a DNA topoisomerase activity that untwists superhelical DNA in an ATP- and Mg2+-dependent reaction. The analogs adenylyl imidodiphosphate and adenyl [beta, gamma-methylene]diphosphonate cannot be used to replace ATP. The topoisomerase activity is not sensitive to the antibiotics oxolinic acid and novobiocin, known antagonists of Escherichia coli DNA gyrase. The possible relationship among the three polypeptides and their biological activities is discussed.
A novel ATP-dependent DNA topoisomerase which makes reversible double-strand breaks in the DNA double helix has been purified to near homogeneity from T4 bacteriophage-infected Escherichia coli cells. Genetic data suggest that this activity is essential for initiating T4 DNA replication forks in vivo.
A detailed physical map of the early region of bacteriophage T7 DNA has been constructed. This map contains: locations for all the cuts made by the restriction endonucleases HindII, HpaII, HaeIII and HaeII, and many of the cuts by HhaI; the approximate end points for each of 61 different deletions; initiation sites and the termination site for RNAs made by Escherichia coli RNA polymerase; an initiation site for RNA made by T7 RNA polymerase; the five primary RNase III cleavage sites of the early region; and the coding sequences for perhaps nine different early proteins. Virtually all of the non-overlapping coding capacity of the five early messenger RNAs is used, except for untranslated stretches of perhaps 30 or so nucleotides at the ends. It seems likely that each of the nine early proteins is made from its own ribosome-binding and initiation site. The mapped restriction cuts provide fixed reference points, and allow DNA fragments containing specific genetic signals to be identified and isolated.
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Phages that have a single-stranded genome, such as ϕX174 and fd, begin their life cycles in infected cells with the conversion of their viral circular DNA into the covalently closed, duplex, parental replicative form (RF) DNA (stage I, single-stranded to double-stranded [SS→DS] DNA replication). The next stage of growth requires a phage-coded protein that will nick the RF DNA at the origin and prepare it as a substrate for further replication by host proteins (stage II, DS→DS DNA replication). In the last stage, phage proteins complex the viral strand, restrict the formation of complementary strands, and allow the assembly of progeny virus containing the viral DNA circle (stage III, DS→SS DNA replication).
Filamentous bacteriophages do not lyse the infected host cell. Their genome is propagated as a “multicopy plasmid” through many bacterial generations, with a concomitant release of phage progeny from the infected host. As a consequence of this parasital
140 independently occurring spontaneous mutations in the lacI gene of Escherichia coli have been examined genetically and physically. DNA sequence analysis of a genetic “hotspot” shows that the tandemly repeating sequence 5′-C-T-G-G-C-T-G-G-C-T-G-G-3′ generates mutations at a high rate, either deleting or adding one unit of four nucleotides (C-T-G-G). Twelve larger deletion mutations have also been sequenced; seven of these were formed by eliminating segments between repeated sequences of five or eight nucleotides, one copy of the repeated sequence remaining after the deletion. Possible mechanisms accounting for the involvement of repeated sequences in the creation of spontaneous mutations are considered.
RESISTANCE to the cytotoxic purine analogue 6-thioguanine (TG) in cultured mammalian cells is known to be associated with deficiency in the X-chromosome-linked purine-salvage enzyme, hypoxanthine-guanine phosphoribosyl transferase (HGPRT, EC 2.4.2.8). The induction of mutations to TG resistance in cultured mammalian cells has been used to quantify the mutagenic effects of various physical and chemical agents1-3 and it has been argued that such mutations arise primarily as a result of true gene mutations4. However, because ionising radiation induces TG-resistant mutants with very low HGPRT activity5 and fails to induce the ouabain-resistance phenotype in mammalian cells6,7, it has been suggested that ionising radiation leads to gross genetic damage rather than to point mutations in structural genes2,7. Here we present evidence that gross structural changes involving the X chromosome are the genetic basis of a significant proportion of radiation-induced mutation to TG resistance in cultured human fibroblasts.
We have isolated and characterized several hundred mutants of bacteriophage λ that are defective in prophage excision. Almost all resemble classical int and xis mutants in complementation pattern and ability to integrate. One int mutant is exceptional in that it is defective in excision but proficient in integration. The mutant sites are located in a region that begins within 50 base-pairs to the right of the attachment site and extends about 1350 base-pairs rightward, int is approximately 1240 base-pairs and xis 110 base-pairs long.
The rate of production of tandem duplications in phage λ has been measured in the presence and absence of known recombination systems. Two deletion phages have been used: tdel33, a deletion derivative of a φ80-λ hybrid phage, and λb221, which carries a large deletion of the central portion of the λ chromosome. Both phages are int−, and tdel33 is also red−, by virtue of their deletions. Stocks of these phages can be prepared free of long tandem duplication derivatives by CsCl density gradient purification. After a single cycle of lytic growth, lysates from these purified phage stocks contain tandem duplications at a frequency of 10−3 in the case of tdel33 and 10−5 in the case of λb221. These frequencies are unaffected by the presence of mutations in the host Rec system or the phage Red system. To investigate the difference in duplication frequency between tdel33 and λb221, the phages were grown in mixed infection. The result indicates that a trans-active product of tdel33 is responsible for its high frequency of duplication production.Tandem duplications have been detected by banding the phage lysates in CsCl density gradients. Long DNA addition mutants can be detected in this way if they arise with a frequency of at least 10−5 and if the duplication length is at least 0.14 λ lengths. To accomplish this it is necessary to distinguish them from contaminating parental phage and from dense phages with aberrant structures which arise at roughly comparable frequencies. The former can be done by rebanding and the latter by growth and rebanding. To distinguish these types we have also made use of a new mutant of Escherichia coli which does not plate λ deletion phages. All of the DNA addition mutants we have detected in this way are tandem duplications; evidently mutants with long insertions arise more rarely.
DNA molecules injected into the macronucleus of Paramecium primaurelia replicate either as free linear telomerized or chromosome integrated molecules. In the present study we show that when a 1.77 kb BamHI DNA fragment harbouring the his3 gene of Saccharomyces cerevisiae was microinjected into the macronucleus, a fraction of the molecules are integrated into the chromosome via an illegitimate recombination process. The injected molecules were mostly inserted at their extremities at multiple points in the genome by replacing the Paramecium sequences. However, insertion sites were not totally at random. Roughly 30% of the molecules were integrated next to or in telomeric repeats. These telomeric repeats were not at the extremities of chromosomes but occupy an internal or interstitial position. We argue that such sites are hotspots for integration as the probability of random insertion near or in an interstitial telomeric site, of which there are 25-60 in a macronucleus is between 5 x 10(-4) and 3 x 10(-5).
Experiments were designed to determine the association between the repair of gamma-radiation-induced DNA double-strand breaks (DSB) and the induction of 700-1000 bp long deletions (Lac(-)----Lac+), base substitutions (leuB19----Leu+), and frameshifts (trpE9777----Trp+) in Escherichia coli K-12. Over the range of 2.5-20 krad, deletions were induced with linear kinetics, as has been shown for the induction of DSB, while the induction kinetics of base substitutions and frameshifts were curvilinear. Like the repair of DSB, deletion induction showed an absolute requirement for an intact recB gene as well as a dependency on the type of preirradiation growth medium; these requirements were not seen for base substitutions or frameshifts. In addition, about 80% of the spontaneous deletions were absent in the recB21 strain. A recC1001 mutation, which confers a 'hyper-Rec' phenotype, increased the rate of gamma-radiation-induced deletions as well as the low-dose production of base substitutions and frameshifts. A recF143 mutation increased the yield of gamma-radiation-induced deletions without increasing base substitutions or frameshifts. A mutS mutation markedly enhanced the gamma-radiation induction of frameshifts, and had a slight effect on base substitutions, but did not affect the induction of deletions. Resistance to gamma-irradiation and the capacity to repair DSB (albeit at about half the normal rate) were restored to the radiosensitive recB21 strain by the addition of the sbcB21 and sbcC201 mutations. However, the radioresistant recB sbcBC strain, which is recombination proficient via the RecF pathway, was still grossly deficient in the ability to produce deletions. A model for deletion induction as a by-product of the recB-dependent (Chi-dependent) repair of gamma-radiation-induced DSB is discussed, as is the inability to detect deletions in cells that use only the recF-dependent (Chi-independent) mechanism to repair DSB.
This report shows that human telomeres are tightly associated with the nuclear matrix. Telomere attachment is observed in several cell types and in all stages of interphase. Mapping experiments show that telomeres are anchored via their TTAGGG repeats; a subtelomeric repeat located immediately proximal to the telomeric TTAGGG repeats is quantitatively released from the nuclear matrix by restriction endonuclease cleavage. TTAGGG repeats introduced at chromosome-internal sites by DNA transfection do not behave as matrix attached loci, suggesting that the telomeric position of the repeats is required for their interaction with the nuclear matrix. These findings are consistent with the idea that telomeres function as a nucleoprotein complex.
To study the mechanism of illegitimate recombination in mammalian cells, we have developed a shuttle vector, pNK1, that contains three bacterial markers, amp (ApR), galK, and neo (KmR). The frequency of deletions occurring in autonomously replicating pNK1 DNA during the growth of monkey COS1 cells was measured by transfecting the plasmid into Escherichia coli cells and counting the number of galK- ApS double mutants among total KmR cells. This method allowed us to test the effects of topoisomerase inhibitors on deletion formation in mammalian cells. The DNA topoisomerase II (TopII) inhibitor, 4'-dimethylepipodophyllotoxin thenylidene-beta-D-glucoside (VM26), stimulated deletions in pNK1 DNA in monkey cells. Since VM26 does not inhibit the strand-break activity of TopII, but rather stabilizes an enzyme-DNA complex in which DNA is cleaved upon treatment with sodium dodecyl sulfate, it is implicated that TopII participates in the deletion process in mammalian cells.
Thirty-seven children and adults who developed acute nonlymphocytic leukemia after the administration of chemotherapy that included etoposide or teniposide for a variety of hematologic and solid malignancies were identified. The secondary leukemia that occurred in these patients could be distinguished from the secondary leukemia that occurs after treatment with alkylating agents by the following: a shorter latency period; a predominance of monocytic or myelomonocytic features; and frequent cytogenetic abnormalities involving 11q23. Patients receiving an epipodophyllotoxin are at risk for developing secondary leukemia that has features distinct from the syndrome of secondary leukemia associated with alkylating agents.
UV irradiation induced the precise excision of Tn10 inserted in met, trp or srl in a Salmonella typhimurium strain; mitomycin C was also found to induce the frequency of precise excision of Tn10 from srl or met. Precise excision of Tn10 was not increased by either UV or mitomycin C in a recA mutant. Similarly, a recA mutant derived from a uvrD strain showed a drastic reduction in the high spontaneous levels of precise excision of Tn10 of this strain. These results indicate that recA is involved in the increased precise excision of Tn10. In contrast to point mutations excision of Tn10 was found to be UV inducible in a top mutant.
Specialized type I topoisomerases catalyze DNA strand transfer during site-specific recombination in prokaryotes and fungi. As a rule, the site specificity of these systems is determined by the DNA binding and cleavage preference of the topoisomerase per se. The Mr 32,000 topoisomerase I encoded by vaccinia virus (a member of the eukaryotic family of "general" type I enzymes) is also selective in its interaction with DNA; binding and cleavage occur in vitro at a pentameric motif 5'-(C or T)CCTT in duplex DNA. Expression of vaccinia virus DNA topoisomerase I in a lambda lysogen of Escherichia coli promotes int-independent excisive recombination of the prophage. To address whether the topoisomerase directly catalyzes DNA strand transfer in vivo, the recombination junctions of plaque-purified progeny phage were cloned and sequenced. In five of six distinct excision events examined, a topoisomerase cleavage sequence is present in one strand of the DNA duplex of both recombining partners. Recombination entails no duplication, insertion, or deletion of nucleotides at the crossover points, consistent with excision via conservative strand exchange at sites of topoisomerase cleavage. Three of these five recombination events are distinguished by the presence of direct repeats at the parental half-sites that extend beyond the pentameric cleavage motif, suggesting that sequence homology may facilitate excision. The data are consistent with a model in which vaccinia topoisomerase catalyzes reciprocal strand transfer, leading to the formation of a nonmigrating Holliday junction, the resolution of which can lead to excisive recombination.
Hepadnaviruses integrate in cellular DNA via an illegitimate recombination mechanism, and clonally propagated integrations are present in most hepatocellular carcinomas which arise in hepadnavirus carriers. Although integration is not specific for any viral or cellular sequence, highly preferred integration sites have been identified near the DR1 and DR2 sequences and in the cohesive overlap region of virion DNA. We have mapped a set of preferred topoisomerase I (Topo I) cleavage sites in the region of DR1 on plus-strand DNA and in the cohesive overlap near DR2 and have tested whether Topo I is capable of mediating illegitimate recombination of woodchuck hepatitis virus (WHV) DNA with cellular DNA by developing an in vitro assay for Topo I-mediated linking. Four in vitro-generated virus-cell hybrid molecules have been cloned, and sequence analysis demonstrated that Topo I can mediate both linkage of WHV DNA to 5'OH acceptor ends of heterologous DNA fragments and linkage of WHV DNA into internal sites of a linear double-stranded cellular DNA. The in vitro integrations occurred at preferred Topo I cleavage sites in WHV DNA adjacent to the DR1 and were nearly identical to a subset of integrations cloned from hepatocellular carcinomas. The end specificity and polarity of viral sequences in the integrations allows us to propose a prototype integration mechanism for both ends of a linearized hepadnavirus DNA molecule.
Cancer may be defined as a progressive series of genetic events that occur in a single clone of cells because of alterations in a limited number of specific genes: the oncogenes and tumor suppressor genes. The association of consistent chromosome aberrations with particular types of cancer has led to the identification of some of these genes and the elucidation of their mechanisms of action. Consistent chromosome aberrations are observed not only in rare tumor types but also in the relatively common lung, colon, and breast cancers. Identification of additional mutated genes through other chromosomal abnormalities will lead to a more complete molecular description of oncogenesis.
Mutations of Escherichia coli K-12 were isolated that increase the frequency of deletion formation. Three of these mutations map to the gene sbcB at 43.5 min on the E. coli chromosome. Two types of mutations at sbcB have been previously defined: sbcB-type that suppress both the UV sensitivity and recombination deficiency of recBC mutants, and xonA-type that suppress only the UV sensitivity. Both types are defective for production of exonuclease I activity. The mutations isolated here were similar to xonA alleles of sbcB because they suppressed the UV sensitivity of recBC mutants but did not restore recombination proficiency. Indeed, two previously characterized xonA alleles were shown to increase the frequency of deletion formation, although an sbcB allele did not. This result demonstrates that loss of exonuclease I activity is not sufficient to confer a high deletion phenotype, rather, the product of the sbcB gene possesses some other function that is important for deletion formation. Because deletion formation in this system is recA independent and does not require extensive DNA homology, these mutations affect a pathway of illegitimate recombination.
A system for detecting a spontaneous deletion in Escherichia coli was developed and the role of DNA gyrase in deletion formation was studied. A derivative of lambda plac5, lambda AM36, was isolated in which whole pBR322 DNA was inserted in the lacZ gene and 227 bp of the lac gene duplicated at both sides of the pBR322 DNA. E. coli lac- strains lysogenized by lambda AM36 had a Lac- phenotype and segregated Lac+ revertants. Sequence analyses showed that the revertant was formed by a deletion that eliminated the inserted pBR322 DNA and one copy of the duplicated segments. The frequency of lac+ revertant formation was independent of recA function, was increased by oxolinic acid, an inhibitor of DNA gyrase, but was not increased in a lysogen of a nalidixic acid-resistant derivative. The reversion frequencies of temperature sensitive mutants of gyrA gene are 10 to 100 times lower than that of the wild-type strain. These results indicate that the DNA gyrase of E. coli participated in the in vivo deletion formation resulting from the direct repeats.
Vaccinia virus encapsidates a Mr 32,000 type IDNA topoisomerase. Although the vaccinia gene encoding the topoisomerase is essential for virus growth, the role of the enzyme in vivo remains unclear. In the present study, the physiologic consequences of vaccinia topoisomerase action have been examined in a heterologous system, Escherichia coli. The vaccinia topoisomerase gene was inducibly expressed in an int-lambda lysogen BL21(DE3) using a T7 RNA polymerase-based transcription system. Expression of active topoisomerase in this context resulted in recA-dependent lysogenic induction as well as cell lysis. Surprisingly, topoisomerase expression also effected a 200-fold increase in the titer of infectious lambda phage, apparently by promoting int-independent prophage excision. This effect was not observed during lysogenic induction with nalidixic acid. Restriction analysis of genomic DNA from plaque-purified excisants revealed (in 10 of 10 cases) gross alterations of the DNA structure around the att site relative to the structure of the parental phage DE3. It is construed therefore that vaccinia DNA topoisomerase I acts to promote illegitimate recombination in E. coli.
A family of A + T-rich sequences termed MARs ("matrix association regions") mediate chromosomal loop attachment. Here we demonstrate that several MARs both specifically bind and contain multiple sites of cleavage by topoisomerase II, a major protein of the mitotic chromosomal scaffold. Interestingly, "hotspots" of enzyme cutting occur within the MAR of the mouse immunoglobulin kappa-chain gene at the breakpoint of a previously described chromosomal translocation. Since topoisomerase II can mediate illegitimate recombination in prokaryotes, we explored further the possibility that MARs might be targets for this process in eukaryotes. We found that a MAR had been deleted from one of the two rabbit immunoglobulin kappa-chain genes and that MARs reside next to a long interspersed repetitive element within the recombination junction of a human ring chromosome 21. These results, taken together with other accounts of nonhomologous recombination, lead to the proposal that a dysfunction of MARs is illegitimate recombination.