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Schematic diagrams of the rolling circle DNA replication assays used to study replication fork movement. Rolling circle replication reactions were performed as described under " Experimental Procedures. " A, standard RCR assay. Reaction mixtures containing DnaB helicase, DNA pol IIIh, ATP, and dNTPs were assembled on ice, and DNA replication was initiated by warming the mixture to 30 °C. In certain instances, DnaB helicase was incubated with 200 M AMP-PNP and TNC template at 30 °C for 5 min to permit binding of DnaB to the single-stranded DNA tail before the addition of DNA pol IIIh, ATP, and dNTPs. B, the RCR assay. The O and P replication proteins and the E. coli DnaJ, DnaK, and GrpE molecular chaperones were used to transfer DnaB helicase onto the tail of the TNC DNA template (in either the presence or absence of SSB) before the initiation of rolling circle DNA replication. For each type of assay, the first round of leading strand DNA synthesis, accompanied by DnaB-mediated displacement of the 5-tail, is pictured. The replication fork apparatus is shown as consisting of a molecule of DnaB helicase (hexameric ring), which translocates 5 3 3 along the linear strand and interacts with a molecule of DNA pol III that is simultaneously synthesizing the growing DNA chain as it moves 3 3 5 along the circular template strand. Newly synthesized DNA is represented by a thicker strand.
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We have used a set of bacteriophage lambda and Escherichia coli replication proteins to establish rolling circle DNA replication in vitro to permit characterization of the functional properties of lambda replication forks. We demonstrate that the lambda replication fork assembly synthesizes leading strand DNA chains at a physiological rate of 650-7...
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... along the strand in the 5 3 3 direction and initiate unwinding of the duplex portion of the TNC DNA template (14). This unwinding mediated by DnaB can be coupled by protein-pro- tein interactions (40) to DNA chain elongation from the 3 terminus of the ()-strand by DNA pol IIIh, producing proces- sive and rapid rolling circle DNA replication ( Fig. 1) ...
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... approaches for assembling DnaB helicase molecules on the 800-base ssDNA tail of the TNC DNA tem- plate, since the relative efficiency of this step might determine the overall efficiency of rolling circle DNA replication on the TNC DNA template. In the standard assay, DnaB is simply mixed with the DNA template in the presence of ATP at 0 °C (Fig. 1A), and DNA synthesis is initiated by the addition of DNA pol IIIh and dNTPs and warming of the reaction mixture to 30 °C. In some experiments, we included 200 M AMP-PNP in the DnaB-binding step, since it has been reported that the binding of DnaB to ssDNA is increased when AMP-PNP is substituted for ATP (41). Although AMP-PNP is ...
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... the helicase activity of DnaB, it does not interfere significantly with the binding of ATP to DnaB (14, 42). Thus, once DnaB is bound to ssDNA in the presence of AMP-PNP, the addition of sufficient quantities of ATP permits helicase func- tion by DnaB and the establishment of the elongation phase of the rolling circle replication reaction (Fig. ...
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... for the transfer of DnaB onto ssDNA or TNC DNA templates involves the use of the O and P proteins (19,20). This multistep transfer reaction is relatively independent of DNA sequence but does require the action of the DnaK/DnaJ/GrpE molecular chaperone system. Additionally, unlike the binding of DnaB alone, the O-and P-mediated transfer reaction (Fig. 1B) is not blocked by stoichiometric coating of ssDNA by SSB (19). In the absence of DnaB, only very modest amounts of DNA synthesis were obtained (about 50 pmol) when the TNC DNA template was incubated with DNA pol IIIh and dNTPs alone (Fig. 2). Extensive DNA synthesis was obtained, however, when DnaB helicase was included along with DNA ...
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... conclude that primase does not affect the strand displacement activity of DNA pol IIIh. SSB stimulated DNA synthesis in the RCR assay when DnaB was absent (Table II, lines 1 and 3), as noted earlier (Fig. 4). On the other hand, in the case of the RCR assay, SSB failed to stimulate DNA synthesis when DnaB was absent (Table II, lines 9 and 11). ...
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... stimulated DNA synthesis in the RCR assay when DnaB was absent (Table II, lines 1 and 3), as noted earlier (Fig. 4). On the other hand, in the case of the RCR assay, SSB failed to stimulate DNA synthesis when DnaB was absent (Table II, lines 9 and 11). Additional studies indicated that the presence of O was responsible for blocking the SSB-mediated stimulation of strand displacement DNA syn- thesis in this situation (data not shown). ...
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... for 15 min at 30 °C and then pro- cessed to determine the level of DNA synthesis. The values listed represent the average of duplicate samples. The average length of the DNA product molecules is calculated by dividing the amount of DNA synthesis by the total amount of template utilized in the assay (0.029 pmol of TNC DNA template; e.g. see Fig. ...
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... TNC template molecules were resolved from un- replicated template molecules by electrophoresis through neu- tral agarose gels. The input TNC template DNA migrated as two distinct subpopulations (Fig. 9, A and B, lane 1). The upper band and the broad smear below it, labeled S in Fig. 9, A and B, apparently consist of nicked circular or gapped circular template molecules with single-stranded DNA tails. The lower band and the faint smear below it (labeled NC in Fig. 9, A and B) apparently are composed of nicked or gapped circular mol- ecules that have ...
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... proportion of TNC template utilized in various rolling circle replication assays was calculated by determining the amount of radiolabeled template remaining at specific time points that comigrated with the input template DNA. The results, depicted in Fig. 10, indicate that a minimum of 60 -70% of the starting template molecules were utilized as substrate within the initial 5 or 10 min of incubation under a variety of RCR assay conditions. This is a minimum estimate for tem- plate utilization, since we have not corrected for the presence of damaged and apparently unproductive tailless ...
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... to this general trend of efficient template utilization was obtained when DNA pol IIIh was the only protein added to the replication assay mixture. In this type of assay, template utilization was very poor, peaking at approximately 10%, con- sistent with the feeble DNA synthesis observed under these conditions (Fig. 3A). The kinetic analysis (Fig. 10) demon- strated that the half-time for template utilization was about 1 min when DNA pol IIIh and SSB were present (open circles) or when DnaB helicase and DNA pol IIIh were included in the RCR assay mixture (open squares). Significantly slower kinet- ics of template utilization were obtained in the latter situation, however, when the ...
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... when DNA pol IIIh and SSB were present (open circles) or when DnaB helicase and DNA pol IIIh were included in the RCR assay mixture (open squares). Significantly slower kinet- ics of template utilization were obtained in the latter situation, however, when the preincubation of DnaB with the TNC tem- plate in the presence of AMP-PNP was omitted (Fig. 10, closed squares). Surprisingly, in the presence of ATP, the O and P replication proteins and the DnaK/DnaJ/GrpE molecular chap- erone group cooperated to load DnaB helicase onto the TNC DNA template and initiate DNA replication somewhat more quickly than when DnaB alone was present ( Fig. 10; compare closed triangles and closed squares). Initiation of ...
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... TNC tem- plate in the presence of AMP-PNP was omitted (Fig. 10, closed squares). Surprisingly, in the presence of ATP, the O and P replication proteins and the DnaK/DnaJ/GrpE molecular chap- erone group cooperated to load DnaB helicase onto the TNC DNA template and initiate DNA replication somewhat more quickly than when DnaB alone was present ( Fig. 10; compare closed triangles and closed squares). Initiation of replication of the TNC template in the RCR system, in fact, lagged only slightly behind the initiation rate found when DnaB helicase was prebound to the ssDNA tail of the TNC template in the presence of AMP-PNP. Finally, we determined that the addi- tion of primase alone, SSB ...
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... values obtained were halved to compensate for the presence of lagging strand DNA synthesis. These values are minimal estimates of the average leading strand chain length, since they have not been corrected for the fact that template utilization was asynchronous and was not completed until about 5 min after initiation of the replication reaction (Fig. 10). The longest leading strand DNA chains, averaging 160 -200 kb, were produced in the RCR assay when SSB was present (Table II, lines 15 and 16). We also found that the presence of SSB or primase in the minimal RCR assay resulted in the synthesis of longer leading strands (Table II, lines ...
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... was anticipated, therefore, that the synthesis of lagging strand DNA chains when primase was present in the RCR assays would lead to an approximate doubling in the level of DNA synthesis. This expectation was met when primase supplemented RCR assays performed in the presence of SSB (Table II, lines 15 and 16). Surprisingly, however, inclusion of primase produced an unexpectedly large 4-fold stimulation of DNA synthesis in the minimal RCR assay when SSB was absent (Table II, lines 5 and 6). ...
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Citations
... Although Pol␥ and mtSSB appear not to interact physically (35), mtSSB can stimulate the primer-extension activity of Pol␥ by imposing an optimal organization of the DNA template (8,36). In other DNA replication systems SSBs have been shown to stimulate the strand displacement activity of DNApols (15,16,(37)(38)(39). However, the effect of mtSSB on the strand displacement activity of Pol␥ , a situation in which the two proteins bind to opposite strands of the DNA fork (Figure 1), remains largely unexplored. ...
Many replicative DNA polymerases couple DNA replication and unwinding activities to perform strand displacement DNA synthesis, a critical ability for DNA metabolism. Strand displacement is tightly regulated by partner proteins, such as single-stranded DNA (ssDNA) binding proteins (SSBs) by a poorly understood mechanism. Here, we use single-molecule optical tweezers and biochemical assays to elucidate the molecular mechanism of strand displacement DNA synthesis by the human mitochondrial DNA polymerase, Polγ, and its modulation by cognate and noncognate SSBs. We show that Polγ exhibits a robust DNA unwinding mechanism, which entails lowering the energy barrier for unwinding of the first base pair of the DNA fork junction, by ∼55%. However, the polymerase cannot prevent the reannealing of the parental strands efficiently, which limits by ∼30-fold its strand displacement activity. We demonstrate that SSBs stimulate the Polγ strand displacement activity through several mechanisms. SSB binding energy to ssDNA additionally increases the destabilization energy at the DNA junction, by ∼25%. Furthermore, SSB interactions with the displaced ssDNA reduce the DNA fork reannealing pressure on Polγ, in turn promoting the productive polymerization state by ∼3-fold. These stimulatory effects are enhanced by species-specific functional interactions and have significant implications in the replication of the human mitochondrial DNA.
... However, the efficiency of Polγ is limited to a few nucleotides [182,[184][185][186][187], in accordance with other DNA polymerases involved in strand displacement synthesis [176,188,189]. Bulk assays have shown that SSBs can stimulate the strand displacement replication of many other DNA polymerases [188,[190][191][192][193], yet the effect of SSB on the strand displacement activity of Polγ remains unexplored. Recently, researchers have used single-molecule manipulation to quantify the effect of cognate and noncognate SSBs on the strand displacement mechanism of Polγ. ...
... Various concentrations of cognate mtSSB and noncognate phage T7 gp2.5 and E. coli SSB have been investigated to examine the potential role of species-specific polymerase-SSB interactions. The study demonstrated [176,177], in accordance with previous findings [188,[190][191][192][193], that SSBs stimulate strand displacement DNA synthesis by utilizing a variety of mechanisms, including the binding of mtSSBs to displaced ssDNA to increase the destabilization energy and reducing the regression pressure on the holoenzyme. These stimulatory effects are also shown to be enhanced by species-specific functional interactions [176,177]. ...
Single-stranded DNA-binding proteins (SSBs) play vital roles in DNA metabolism. Proteins of the SSB family exclusively and transiently bind to ssDNA, preventing the DNA double helix from re-annealing and maintaining genome integrity. In the meantime, they interact and coordinate with various proteins vital for DNA replication, recombination, and repair. Although SSB is essential for DNA metabolism, proteins of the SSB family have been long described as accessory players, primarily due to their unclear dynamics and mechanistic interaction with DNA and its partners. Recently-developed single-molecule tools, together with biochemical ensemble techniques and structural methods, have enhanced our understanding of the different coordination roles that SSB plays during DNA metabolism. In this review, we discuss how single-molecule assays, such as optical tweezers, magnetic tweezers, Förster resonance energy transfer, and their combinations, have advanced our understanding of the binding dynamics of SSBs to ssDNA and their interaction with other proteins partners. We highlight the central coordination role that the SSB protein plays by directly modulating other proteins’ activities, rather than as an accessory player. Many possible modes of SSB interaction with protein partners are discussed, which together provide a bigger picture of the interaction network shaped by SSB.
... This is in reasonable agreement with the historical 770 nt.s -1 rate estimated 440 from the 100 minutes required to transfer the whole 4.6 Mb E. coli chromosome (Jacob and Wollman, 441 1958). Besides, the rate of DNA synthesis by the DNA polymerase III holoenzyme during RCR was 442 estimated at 650-750 nuc.s -1(Stephens and McMacken, 1997). By comparison, the rate of TraI 443 helicase activity was measured at 1120 ± 160 bp.s -1(Sikora et al., 2006). ...
DNA conjugation is a contact-dependent horizontal gene transfer mechanism responsible for disseminating drug resistance among bacterial species. Conjugation remains poorly characterised at the cellular scale, particularly regarding the reactions occurring after the plasmid enters the new host cell. Here, we use live-cell microscopy to visualise the intracellular dynamics of conjugation in real time. We reveal that the transfer of the plasmid in single-stranded DNA (ssDNA) form followed by its conversion into double-stranded DNA (dsDNA) are fast and efficient processes that occur with specific timing and subcellular localisation. Notably, the ss-to-dsDNA conversion is the critical step that governs the timing of plasmid-encoded protein production. The leading region that first enters the recipient cell carries single-stranded promoters that allow the early and transient synthesis of leading proteins immediately upon entry of the ssDNA plasmid. The subsequent ss-to-dsDNA conversion turns off leading gene expression and licences the expression of the other plasmid genes under the control of conventional double-stranded promoters. This elegant molecular strategy evolved by the conjugative plasmid allows for the timely production of factors sequentially involved in establishing, maintaining and disseminating the plasmid.
... Although physical interactions between 3 Poland mtSSB have not been observed (41), mtSSB can stimulate the primer-extension activity of Pol (17,42). In other DNA replication systems SSBs have been shown to stimulate the strand displacement activity of DNApols (13,14,33,43,44). However, the effect of mtSSB on the strand displacement activity of Pol remains largely unexplored. ...
Many replicative DNA polymerases couple DNA replication and unwinding activities to perform strand displacement DNA synthesis, a critical ability for DNA metabolism. Strand displacement is tightly regulated by partner proteins, such as single-stranded DNA (ssDNA) binding proteins (SSBs) by a poorly understood mechanism. Here, we use single-molecule optical tweezers and biochemical assays to elucidate the molecular mechanism of strand displacement DNA synthesis by the human mitochondrial DNA polymerase, Polγ, and its modulation by cognate and noncognate SSBs. We show that Polγ exhibits a robust DNA unwinding mechanism, which entails lowering the energy barrier for unwinding of the first base pair of the DNA fork junction, by ~55%. However, the polymerase cannot prevent the reannealing of the parental strands efficiently, which limits by ~30-fold its strand displacement activity. We demonstrate that SSBs stimulate the Polγ strand displacement activity through several mechanisms. SSB binding energy to ssDNA additionally increases the destabilization energy at the DNA junction, by ~25%. Furthermore, SSB interactions with the displaced ssDNA reduce the DNA fork reannealing pressure on Polγ, in turn promoting the productive polymerization state by ~3-fold. These stimulatory effects are enhanced by species-specific functional interactions and have significant implications in the replication of the human mitochondrial DNA.
... The AT-rich region is followed by inverted repeats capable of forming a stemloop structure similar to the one found in phage Lc-Nu (80). J-1 gp41 (gp37 in PL-1) is similar to DnaB helicases (81), and gp43 (gp39 in PL-1) is a putative DNA binding protein. J-1 gp44 (gp40 in PL-1) is a putative RusA-like Holliday junction resolvase (82). ...
... This late-phase DNA replication, which is believed to be independent of an origin of replication, has been reconstituted in vitro for viruses that encode their own polymerase [e.g. HSV-1, T7, T4 and È29, (11)(12)(13)(14)] and for bacteriophage (15). ...
... , like SPP1, encodes an origin-specific initiation protein (O protein) that is not a DnaA homolog. The O protein, like SPP1 G38P, can serve to initiate DNA synthesis on artificial replication forks that lack origin sequences (15). SV40, like SPP1, encodes its own origin binding protein and helicase, both contained within the same polypeptide chain as the T-antigen (52). ...
Complex viruses that encode their own initiation proteins and subvert the host’s elongation apparatus have provided valuable
insights into DNA replication. Using purified bacteriophage SPP1 and Bacillus subtilis proteins, we have reconstituted a rolling circle replication system that recapitulates genetically defined protein requirements.
Eleven proteins are required: phage-encoded helicase (G40P), helicase loader (G39P), origin binding protein (G38P) and G36P single-stranded DNA-binding protein (SSB); and host-encoded PolC and DnaE polymerases, processivity factor (β2), clamp loader (τ-δ-δ′) and primase (DnaG). This study revealed a new role for the SPP1 origin binding protein. In the presence of SSB, it is required for initiation
on replication forks that lack origin sequences, mimicking the activity of the PriA replication restart protein in bacteria.
The SPP1 replisome is supported by both host and viral SSBs, but phage SSB is unable to support B. subtilis replication, likely owing to its inability to stimulate the PolC holoenzyme in the B. subtilis context. Moreover, phage SSB inhibits host replication, defining a new mechanism by which bacterial replication could be
regulated by a viral factor.
... The loading of DnaB onto ssDNA, formed by strand separation within the high-AT-rich region of oril, was suggested to mark the end of the initiation phase of l theta mode DNA replication[55]. Previously, we confirmed that theta-mode oril-dependent prophage replication initiation, which requires P interaction with, and loading of, DnaB, was inhibited if the host carried the dnaBGrpD55 mutation, yet there was no obvious influence of this allele on E. coli DNA propagation[18]. ...
Several earlier studies have described an unusual exclusion phenotype exhibited by cells with plasmids carrying a portion of the replication region of phage lambda. Cells exhibiting this inhibition phenotype (IP) prevent the plating of homo-immune and hybrid hetero-immune lambdoid phages. We have attempted to define aspects of IP, and show that it is directed to repλ phages. IP was observed in cells with plasmids containing a λ DNA fragment including oop, encoding a short OOP micro RNA, and part of the lambda origin of replication, oriλ, defined by iteron sequences ITN1-4 and an adjacent high AT-rich sequence. Transcription of the intact oop sequence from its promoter, pO is required for IP, as are iterons ITN3–4, but not the high AT-rich portion of oriλ. The results suggest that IP silencing is directed to theta mode replication initiation from an infecting repλ genome, or an induced repλ prophage. Phage mutations suppressing IP, i.e., Sip, map within, or adjacent to cro or in O, or both. Our results for plasmid based IP suggest the hypothesis that there is a natural mechanism for silencing early theta-mode replication initiation, i.e. the buildup of λ genomes with oop+
oriλ+ sequence.
... For several decades, E. coli has provided the prototype for biochemical understanding of the replication of a cellular chromosome (Baker and Kornberg, 1991;Marians, 2000). Our mechanistic knowledge has been facilitated, and often led, by biochemical studies in complete systems encoded by bacteriophages l, T4, T7, F29, and SV40 (Alberts, 1987;Benkovic et al., 2001;Collins et al., 1993;Nossal et al., 2007;Richardson, 1983;Salas et al., 1995;Stephens and McMacken, 1997;Waga and Stillman, 1994;Wang et al., 2000). The viral SV40-encoded origin recognition, helicase loader, and helicase activities of the multifunctional T-antigen have provided significant knowledge related to eukaryotic replication, yet no system is available for the reconstitution of cellular eukaryotic replication forks with purified proteins. ...
We have expressed and purified 13 proteins predicted to be required for B. subtilis DNA replication. When combined with a circular DNA template with a 5' unpaired flap, these proteins reconstitute replication of both the leading and lagging strands at the physiological rate. Consistent with the in vivo requirement for two DNA polymerase III replicases for B. subtilis chromosomal replication, both PolC and DnaE are required for reconstitution of the replication fork in vitro. Leading strand synthesis requires PolC plus ten proteins; lagging strand synthesis additionally requires primase and DnaE. DnaE does not serve as the lagging strand replicase, like DNA polymerase delta in eukaryotes, but instead functions like eukaryotic DNA polymerase alpha, adding a stretch of deoxynucleotides to the RNA primer before handoff to PolC. Primase equilibrates with the fork prior to synthesis of each Okazaki fragment, and its concentration controls the frequency of initiation and Okazaki fragment size.
... delbrueckii phage LL-H. E. coli DnaC and lambda P protein are known to recruit the bacterial DnaB replicative helicase to the initiator complexes assembled at the origin of replication and to promote the formation of the replication fork (Stephens and McMacken, 1997). An AT-rich sequence, comprised of five short direct repeats, is present in two or three copies in AAT3 ORF31 (the potential DnaC protein) and ORF32. ...
The complete genomic sequence of a temperate bacteriophage PhiAT3 isolated from Lactobacillus (Lb.) casei ATCC 393 is reported. The phage consists of a linear DNA genome of 39,166 bp, an isometric head of 53 nm in diameter, and a flexible, noncontractile tail of approximately 200 nm in length. The number of potential open reading frames on the phage genome is 53. There are 15 unpaired nucleotides at both 5' ends of the PhiAT3 genome, indicating that the phage uses a cos-site for DNA packaging. The PhiAT3 genome was grouped into five distinct functional clusters: DNA packaging, morphogenesis, lysis, lysogenic/lytic switch, and replication. The amino acid sequences at the NH2-termini of some major proteins were determined. An in vivo integration assay for the PhiAT3 integrase (Int) protein in several lactobacilli was conducted by constructing an integration vector including PhiAT3 int and the attP (int-attP) region. It was found that PhiAT3 integrated at the tRNAArg gene locus of Lactobacillus rhamnosus HN 001, similar to that observed in its native host, Lb. casei ATCC 393.
... l O(am)8 phage has the amber mutation in gene O encoding the l O protein which is responsible for initiation of phage DNA replication. Lack of l O(am)8 replication in the 594 sup + host was first shown by Ogawa & Tomizawa (1968) and Shuster & Weisbach (1969) and the role of the gene O product in initiation of phage DNA replication is now well established (Wyatt & Inokuchi, 1974;Zylicz et al., 1984;Dodson et al., 1989;Stephens & McMacken, 1997). The absence of l O(am)8 phage DNA replication in the 594 sup + host is also supported by our experiments with mutants 594 sup + dnaQ49 and 594 sup + mutL. ...
... It may be seen in Table 2 that when l O(am)8 phage is not irradiated with UV light, the level of l O + revertants is the same in nonirradiated (-SOS) and irradiated (+SOS) 594 sup + cells. This is in agreement with in vitro studies on the crucial role of the l O protein in initiation of l phage DNA replication (Zylicz et al., 1984;Stephens & McMacken, 1997). This is good evidence that DNA replication of l O(am)8 phage in the 594 sup + host does not occur, and strongly suggests that the observed UV-and MMS-induced mutations of l O(am)8 phage in 594 sup + cells are generated before initiation of DNA replication. ...
Mutagenesis in Escherichia coli, a subject of many years of study is considered to be related to DNA replication. DNA lesions nonrepaired by the error-free nucleotide excision repair (NER), base excision repair (BER) and recombination repair (RR), stop replication at the fork. Reinitiation needs translesion synthesis (TLS) by DNA polymerase V (UmuC), which in the presence of accessory proteins, UmuD', RecA and ssDNA-binding protein (SSB), has an ability to bypass the lesion with high mutagenicity. This enables reinitiation and extension of DNA replication by DNA polymerase III (Pol III). We studied UV- and MMS-induced mutagenesis of lambdaO(am)8 phage in E. coli 594 sup+ host, unable to replicate the phage DNA, as a possible model for mutagenesis induced in nondividing cells (e.g. somatic cells). We show that in E. coli 594 sup+ cells UV- and MMS-induced mutagenesis of lambdaO(am)8 phage may occur. This mutagenic process requires both the UmuD' and C proteins, albeit a high level of UmuD' and low level of UmuC seem to be necessary and sufficient. We compared UV-induced mutagenesis of lambdaO(am)8 in nonpermissive (594 sup+) and permissive (C600 supE) conditions for phage DNA replication. It appeared that while the mutagenesis of lambdaO(am)8 in 594 sup+ requires the UmuD' and C proteins, which can not be replaced by other SOS-inducible protein(s), in C600 supE their functions may be replaced by other inducible protein(s), possibly DNA polymerase IV (DinB). Mutations induced under nonpermissive conditions for phage DNA replication are resistant to mismatch repair (MMR), while among those induced under permissive conditions, only about 40% are resistant.
