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1,2-dihydro-2-oxoadenine (2-OH-A), a common DNA lesion produced by reactive oxygen species, is a strong replicative block
for several DNA polymerases (DNA pols). We have previously shown that various bases can be misincorporated opposite the 2-OH-A
lesion and the type of mispairs varies with either the sequence context or the type of DNA pol tested...
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Proliferating cell nuclear antigen (PCNA) has been shown to interact with a variety of DNA polymerases (pol) such as pol delta, pol epsilon, pol iota, pol kappa, pol eta, and pol beta. Here we show that PCNA directly interacts with the newly discovered pol lambda cloned from human cells. This interaction stabilizes the binding of pol lambda to the...
In this paper we show that DNA polymerase lambda (pol lambda) interacts with proliferating cell nuclear antigen (PCNA) in vivo in human cells. Moreover, by using recombinant mutated PCNA, we could demonstrate that pol lambda interacts with both the interdomain-connecting loop and the nearby hydrophobic pocket on the anterior of PCNA and that critic...
Human replication protein A (huRPA) is a multisubunit protein which is involved in DNA replication, repair and recombination
processes. It exists as a stable heterotrimer consisting of p70, p32 and p14 subunits. To understand the contribution of huRPA
subunits to DNA binding we applied the photoaffinity labeling technique. The photoreactive oligonu...
DNA polymerase (pol) ε is thought to be the leading strand replicase in eukaryotes, whereas pols λ and β are believed to be mainly involved in re-synthesis steps of DNA repair. DNA elongation by the human pol ε is halted by an abasic site (apurinic/apyrimidinic (AP) site). In this study, we present in vitro evidence that human pols λ, β, and η can...
Apurinic/apyrimidinic (AP) sites are continuously generated in genomic DNA. Left unrepaired, AP sites represent noninstructional premutagenic lesions that are impediments to DNA synthesis. When DNA polymerases encounter an AP site, they generally insert dAMP. This preferential insertion is referred to as the A-rule. Crystallographic structures of D...
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... In later experiments, a technically less complicated approach was used, whereby a primer extension assay with four parallel DNA polymerase reactions was carried out simultaneously (each of them with only one of four dNTPs) was usually performed to ascertain which nucleotide is incorporated opposite a certain lesion [9][10][11]. However, in all these experiments, the primer extension reaction products were not separated by electrophoresis, and therefore no conclusion could be drawn concerning the question if these lesions cause the misincorporation of only one type of nucleotides and don't generate other mutations as well. ...
We propose an improved earlier described “mirror” method for detecting in cell nuclear extracts mutations that arise in DNA during its replication due to the misincorporation of deoxyadenosine-5′-monophosphate (dAMP) opposite 7,8-dihydro-8-oxoguanine (8-oxoG). This method is based on the synthesis of a complementary chain (“mirror”) by nuclear extracts of different mice organs on a template containing 8-oxoG and dideoxycytidine residue (ddC) at the 3′‑end. The “mirror” was amplified by PCR using primers part of which was non-complementary to the template. It allowed obtaining the “framed mirror” products. The misincorporation of dAMP in “framed mirror” products forms an EcoRI restriction site. The restriction analysis of double-stranded “framed mirror” products allows a quantification of the mutation frequency in nuclear extracts. The data obtained show that the mutagenic potential of 8-oxoG markedly varied in different organs of adult mice and embryos.
... POLλ interacts with OGG1, SMUG1 and MPG, suggesting its role as a POLβ backup polymerase/dRPase [175,176]. Aside from that, POLλ efficiently and accurately bypasses 2hydroxyadenine and oxoG lesions and physically interacts with MUTYH DNA glycosylase, suggesting its possible involvement in OGG1/MUTYH-dependent faithful repair of oxidized purines [177][178][179]. ...
Base excision DNA repair (BER) is a vitally important pathway that protects the cell genome from many kinds of DNA damage, including oxidation, deamination, and hydrolysis. It involves several tightly coordinated steps, starting from damaged base excision and followed by nicking one DNA strand, incorporating an undamaged nucleotide, and DNA ligation. Deficiencies in BER are often embryonic lethal or cause morbid diseases such as cancer, neurodegeneration, or severe immune pathologies. Starting from the early 1980s, when the first mammalian cell lines lacking BER were produced by spontaneous mutagenesis, such lines have become a treasure trove of valuable information about the mechanisms of BER, often revealing unexpected connections with other cellular processes, such as antibody maturation or epigenetic demethylation. In addition, these cell lines have found an increasing use in genotoxicity testing, where they provide increased sensitivity and representativity to cell-based assay panels. In this review, we outline current knowledge about BER-deficient cell lines and their use.
... Having electrophoretically separated the reaction products, they determined the potentially mutagenic incorporation of deoxyadenosine-5 -monophosphate (dAMP) as the sole possible misincorporation opposite 8-oxoG. Today, the primer extension assay, with four parallel DNA-dependent polymerase reactions carried out simultaneously (each of them with only one of the four types of nucleoside triphosphate (dNTP)), is usually performed in order to determine which nucleotide is incorporated opposite a certain lesion [7][8][9]. However, in all of those experiments, no separation of the primer extension reaction products was performed, and therefore no conclusion can be drawn as to whether those lesions cause the misincorporation of only one type of a nucleotide and do not generate other mutations as well. ...
We propose an improved method for detecting mutations that arise in DNA due to misincorporations of deoxyadenosine-5′-monophosphate (dAMP) opposite 7,8-dihydro-8-oxoguanine (8-oxoG). The method is based on the synthesis of complementary chains (“mirror” products) using a template containing 8-oxoG. The misincorporation of dAMP in the “mirror” product forms EcoRI sites. The restriction analysis of double-stranded DNAs obtained by PCR of “mirror” product allows quantification of the mutagenesis frequency. In addition, single-strand conformational polymorphism (SSCP) analysis of the single-stranded “mirror” products shows that different DNA polymerases only incorporate dA or dC opposite 8-oxoG. The proposed approach used in developing this technique can be applied in the study of other lesions as well, both single and clustered.
... [15][16][17][18][19] Furthermore, Polλ may also function in DNA damage tolerance as it is capable of efficiently performing translesion synthesis (TLS) on DNA substrates that contain various different lesions. [20][21][22][23][24][25] However, the manner in which a specific DNA polymerase, such as Polλ, is recruited for each of these DNA damage repair and tolerance pathways is not well established. ...
... Importantly, there were not significant differences in survival of cells transiently expressing wild-type Polλ, the deletion mutants of Polλ, or an unmodified plasmid in the absence of DNA damaging treatment ( Figure S8). [11][12][13][14][15][16][17][18][19][20][21][22][23][24][25] It has been proposed that the unique N-terminal domains of Polλ may be involved in regulating its participation in these responses to DNA damage. Considering that the primary BER polymerase, Polβ possesses only the catalytic domains (Figure 1), it is unclear whether the N-terminal domains of Polλ contribute to its involvement in BER. ...
Specialized DNA polymerases, such as DNA polymerase lambda (Polλ), are important players in DNA damage tolerance and repair pathways. Knowing how DNA polymerases are regulated and recruited to sites of DNA damage is imperative to understanding these pathways. Recent work has suggested that Polλ plays a role in several distinct DNA damage tolerance and repair pathways. In this paper, we report previously unknown roles of the N-terminal domains of human Polλ for modulating its involvement in DNA damage tolerance and repair. Using Western blot analysis, fluorescence microscopy, and cell survival assays, we found that the BRCA1 C-terminal (BRCT) and proline/serine-rich (PSR) domains of Polλ affect its cellular localization and DNA damage responses. The nuclear localization signal (NLS) of Polλ was necessary to overcome the impediment of its nuclear localization caused by its BRCT and PSR domains. Induction of DNA damage resulted in recruitment of Polλ to chromatin, which was controlled by its BRCT and PSR domains. In addition, the presence of both domains was required for Polλ-mediated tolerance of oxidative DNA damage but not DNA methylation damage. These findings suggest that the N-terminal domains of Polλ are important for regulating its responses to DNA damage.
... More recent studies showed that the effect is especially great when a damaged template is used in synthesis. DNA polymerase λ is capable of synthesizing DNA on a template containing 8-oxoguanine or 1,2-dihydro-2-oxoadenine lesions, but the fidelity of this synthesis is low; i.e., the probability of adding a proper nucleotide (dCTP and or dTTP, respectively) is comparable with that of adding a mismatch (dATP or dGTP, respectively) [68,69]. RPA substantially reduces the erroneous dATP incorporation rate on 8-oxoguanine-containing templates. ...
... The proliferating cell nuclear antigen (PCNA) added to the mixture together with RPA facilitates dCTP incorporation, increasing the probability of correct nucleotide addition by DNA polymerase λ by a factor of 1200 [68]. When the template contains the other lesion (1,2-dihydro-2oxoadenine), PCNA and RPA acting together increase the probability of correct dTTP incorporation by a factor of 166 [69]. Thus, RPA and PCNA are important components of a functional replication complex and affect not only major replicative DNA polymerases (α and δ), but also specialized polymerases that synthesize DNA in a replication fork blocked by a lesion in the template strand. ...
Replication protein A (RPA) is a key regulator of eukaryotic DNA metabolism. RPA is a highly conserved heterotrimeric protein and contains multiple oligonucleotide/oligosaccharide-binding folds. The major RPA function is binding to single-stranded DNA (ssDNA) intermediates forming in DNA replication, repair, and recombination. Although binding ssDNA with high affinity, RPA can rapidly diffuse along ssDNA and destabilizes the DNA secondary structure. A highly dynamic RPA binding to ssDNA allows other proteins to access ssDNA and to displace RPA from the RPA–ssDNA complex. As has been shown recently, RPA in complex with ssDNA is posttranslationally modified in response to DNA damage. These modifications modulate the RPA interactions with its protein partners and control the DNA damage signaling pathways. The review considers up-to-date data on the RPA function as an active coordinator of ssDNA intermediate processing within DNA metabolic pathways, DNA repair in particular.
... Extensive studies have implicated crucial role of Pol λ in mammalian base excision repair, error-free translesion DNA synthesis in oxidative DNA damage [11][12] and repair of double strand breaks (DSBs) via non-homologous end joining (NHEJ) pathway [13][14][15][16]. In addition, recent studies have indicated specific requirement of Pol λ in cell cycle progression in response to oxidative damage and its functional link to the S phase specific DNA damage response machinery in cancer cells [17]. ...
Here, we have investigated the physical and molecular basis of stability of Arabidopsis DNA Pol λ, the sole X family DNA polymerase member in plant genome, under UV-B and salinity stress in connection with the function of the N-terminal BRCT (breast cancer-associated C terminus) domain and Ser-Pro rich region in the regulation of the overall structure of this protein. Tryptophan fluorescence studies, fluorescence quenching and Bis-ANS binding experiments using purified recombinant full length Pol λ and its N-terminal deletion forms have revealed UV-B induced conformational change in BRCT domain deficient Pol λ. On the other hand, the highly conserved C-terminal catalytic core PolX domain maintained its ter-tiary folds under similar condition. Circular dichroism (CD) and fourier transform infrared (FT-IR) spectral studies have indicated appreciable change in the secondary structural elements in UV-B exposed BRCT domain deficient Pol λ. Increased thermodynamic stability of the C-terminal catalytic core domain suggested destabilizing effect of the N-terminal Ser-Pro rich region on the protein structure. Urea-induced equilibrium unfolding studies have revealed increased stability of Pol λ and its N-terminal deletion mutants at high NaCl concentration. In vivo aggregation studies using transient expression systems in Arabidopsis and tobacco indicated possible aggregation of Pol λ lacking the BRCT domain. Immunopre-cipitation assays revealed interaction of Pol λ with the eukaryotic molecular chaperone HSP90, suggesting the possibility of regulation of Pol λ stability by HSP90 in plant cell. Overall, our results have provided one of the first comprehensive information on the bio-physical characteristics of Pol λ and indicated the importance of both BRCT and Ser-Pro rich modules in regulating the stability of this protein under genotoxic stress in plants.
... Extensive studies have implicated crucial role of Pol λ in mammalian base excision repair, error-free translesion DNA synthesis in oxidative DNA damage [11][12] and repair of double strand breaks (DSBs) via non-homologous end joining (NHEJ) pathway [13][14][15][16]. In addition, recent studies have indicated specific requirement of Pol λ in cell cycle progression in response to oxidative damage and its functional link to the S phase specific DNA damage response machinery in cancer cells [17]. ...
Here, we have investigated the physical and molecular basis of stability of Arabidopsis DNA
Pol λ, the sole X family DNA polymerase member in plant genome, under UV-B and salinity
stress in connection with the function of the N-terminal BRCT (breast cancer-associated C
terminus) domain and Ser-Pro rich region in the regulation of the overall structure of this protein.
Tryptophan fluorescence studies, fluorescence quenching and Bis-ANS binding experiments
using purified recombinant full length Pol λ and its N-terminal deletion forms have
revealed UV-B induced conformational change in BRCT domain deficient Pol λ. On the
other hand, the highly conserved C-terminal catalytic core PolX domain maintained its tertiary
folds under similar condition. Circular dichroism (CD) and fourier transform infrared
(FT-IR) spectral studies have indicated appreciable change in the secondary structural elements
in UV-B exposed BRCT domain deficient Pol λ. Increased thermodynamic stability of
the C-terminal catalytic core domain suggested destabilizing effect of the N-terminal Ser-
Pro rich region on the protein structure. Urea-induced equilibrium unfolding studies have
revealed increased stability of Pol λ and its N-terminal deletion mutants at high NaCl concentration.
In vivo aggregation studies using transient expression systems in Arabidopsis
and tobacco indicated possible aggregation of Pol λ lacking the BRCT domain. Immunoprecipitation
assays revealed interaction of Pol λ with the eukaryotic molecular chaperone
HSP90, suggesting the possibility of regulation of Pol λ stability by HSP90 in plant cell.
Overall, our results have provided one of the first comprehensive information on the biophysical
characteristics of Pol λ and indicated the importance of both BRCT and Ser-Pro
rich modules in regulating the stability of this protein under genotoxic stress in plants.
... Since DNA polymerase  lacks its own proofreading 3 -5 exonuclease activity and has relatively low fidelity, it can be assumed to utilize damaged DNA templates in DNA synthesis. Experiments in vitro showed that DNA polymerase  is capable of catalyzing DNA synthesis through reactive oxygen species-induced lesions, such as 7,8-dihydro-8-oxoguanine (8oxoG) [77], 2-hydroxyadenine (2-OH-A) [78], 2-deoxyribonolactone [79] and AP sites [80][81][82]; UV irradiation-induced lesions, such as (64)TT photoproducts and cyclobutane dimers (CPDs) [83]; and alkylation lesions, such as O 6 -methylguanine [84] or lesions induced by certain anticancer drugs, including cisplatin [85][86][87][88][89]. The role of DNA polymerase  in translesion DNA synthesis is considered in more detail below. ...
... Since DNA polymerase forms only a few contacts with the template DNA strand when interacting with the DNA duplex [103] and, on the other hand, is capable of utilizing DNA substrates with a limited complementarity in the primer-template duplex part [54], the enzyme is likely to catalyze translesion DNA synthesis. This activity of DNA polymerase was indeed observed in vitro on substrates containing various lesions, such as AP sites [80,104], 8oxoG [105,106], 2-OH-A [78], and platinum adducts [104]. The role of DNA polymerase in translesion DNA synthesis is detailed below. ...
... As it was found recently, DNA polymerases  and are capable of synthesizing DNA not only through AP sites, but also through other lesions induced by reactive oxygen species. Such lesions include 8oxoG and 2-OH-A [77,78,105,106,127,178,179]. The mutagenic potential of 8oxoG is well known. ...
Among the set of mammalian DNA polymerases, DNA polymerases belonging to the X and Y families have a special place. The majority of these enzymes are involved in repair, including base excision repair and non-homologous end joining. Some of them play a crucial role during the specific process which is referred to as translesion synthesis (TLS). TLS intends for the cell surviving during the replication of damaged DNA templates. Additionally, specific activities of TLS-polymerases have to be useful for repair of double-stranded clustered lesions: if the synthesis is proceeded via base excision repair process, the role of DNA polymerases β or λ will be important. In this review we discussed the biochemical properties and functional relevance of X family DNA polymerases β and λ.
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... 9 In recent years, interest in DNA Pol λ has been increased significantly by the observation that the error-free lesions bypass activity of this protein for oxidative DNA damage enhanced strongly by the auxiliary factors PCNA and RPA. 10 In contrast, members of Y family DNA Pols have been shown to be involved in error-prone translesion DNA synthesis. 11 Our recent study has demonstrated significant levels of induction of Pol λ transcript in Arabidopsis seedlings following UV-B exposure and the involvement of Pol λ protein in gapfilling DNA synthesis in NER during removal of UV-B-induced DNA lesion in Arabidopsis. 2 Participation of Pol λ has also been demonstrated in highly ...
DNA repair mechanisms are essential for the maintenance of genomic stability, proper cellular function and survival for all organisms. Plants, with their intrinsic immobility, are vastly exposed to a wide range of environmental agents and also endogenous processes which frequently cause damage to DNA and impose genotoxic stress. Therefore, in order to survive under frequent and extreme environmental stress conditions, plants have developed a vast array of efficient and powerful DNA damage repair mechanisms to ensure rapid and precise repair of genetic material for maintaining genome stability and faithful transfer of genetic information over generations. ( 1) Recently, we have defined the role of DNA polymerase λ in repair of UV-B-induced photoproducts in Arabidopsis thaliana via nucleotide excision repair pathway. ( 2) Here, we have further discussed potential function of DNA polymerase λ in various DNA repair pathways in higher plant genome in response to environmental and genotoxic stress factors.
... Our previous results showed that DNA pol is the most proficient, among human DNA pols, in performing error-free bypass of 2-OH-A and 8-oxo-G lesions. This feature is essential both for TLS at the fork and for correct repair of the A:8-oxo-G mismatches (8)(9)(10). Finally, a convincing set of data exists, suggesting to a role of DNA pol in NHEJ-dependent repair of ds DNA breaks (DSBs) (11). ...
Human DNA polymerase (pol) λ functions in base excision repair and non-homologous end joining. We have previously shown that DNA pol λ is involved in accurate bypass of the two frequent oxidative lesions, 7,8-dihydro-8-oxoguanine and 1,2-dihydro-2-oxoadenine during the S phase. However, nothing is known so far about the relationship of DNA pol λ with the S phase DNA damage response checkpoint. Here, we show that a knockdown of DNA pol λ, but not of its close homologue DNA pol β, results in replication fork stress and activates the S phase checkpoint, slowing S phase progression in different human cancer cell lines. We furthermore show that DNA pol λ protects cells from oxidative DNA damage and also functions in rescuing stalled replication forks. Its absence becomes lethal for a cell when a functional checkpoint is missing, suggesting a DNA synthesis deficiency. Our results provide the first evidence, to our knowledge, that DNA pol λ is required for cell cycle progression and is functionally connected to the S phase DNA damage response machinery in cancer cells.
