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In bacterial cells, DNA damage tolerance is manifested by the action of translesion DNA polymerases that can synthesize DNA across template lesions that typically block the replicative DNA polymerase III. It has been suggested that one of these TLS DNA polymerases, DNA polymerase IV, can either act in concert with the replisome, switching places on the β sliding clamp with DNA polymerase III to bypass the template damage, or act subsequent to the replisome skipping over the template lesion in the gap in nascent DNA left behind as the replisome continues downstream. Evidence exists in support of both mechanisms. Using single-molecule analyses we show that DNA polymerase IV associates with the replisome in a concentration-dependent manner and remains associated over long stretches of replication fork progression under unstressed conditions. This association slows the replisome, requires DNA polymerase IV binding to the β clamp but not its catalytic activity, and is reinforced by the presence of the γ subunit of the β clamp-loading DnaX complex in the DNA polymerase III holoenzyme. Thus, DNA damage is not required for association of DNA polymerase IV with the replisome. We suggest that under stress conditions such as induction of the SOS response, the association of DNA polymerase IV with the replisome provides both a surveillance/bypass mechanism and a means to slow replication fork progression, thereby reducing the frequency of collisions with template damage and the overall mutagenic potential.SignificanceDamage to the nucleotide bases that make up the DNA in chromosomes creates a problem for their subsequent accurate duplication each time a cell divides. Typically, the cellular enzymatic machinery that replicates the DNA cannot copy a damaged base and specialized trans-lesion DNA polymerases, which are prone to making errors that result in mutations, are required to copy the damaged base, allowing replication to proceed. We demonstrate that the bacterial replisome, which is comprised of the enzymes required to replicate the chromosome, can associate with one of these specialized trans-lesion polymerases over long distances of replicated DNA. This association slows the speed of replication, thereby reducing the chance of mutations arising in the cell under conditions of stress.

Abasic sites are among the most abundant DNA lesions encountered by cells. Their replication requires actions of specialized DNA polymerases. Herein, two archaeal specialized DNA polymerases were examined for their capability to perform translesion DNA synthesis (TLS) on the lesion, including Sulfolobuss islandicus Dpo2 of B-family, and Dpo4 of Y-family. We found neither Dpo2 nor Dpo4 is efficient to complete abasic sites bypass alone, but their sequential actions promote lesion bypass. Enzyme kinetics studies further revealed that the Dpo4’s activity is significantly inhibited at +1 to +3 site past the lesion, at which Dpo2 efficiently extends the primer termini. Furthermore, their activities are inhibited upon synthesis of 5–6 nt TLS patches. Once handed over to Dpo1, these substrates basically inactivate its exonuclease, enabling the transition from proofreading to polymerization of the replicase. Collectively, by functioning as an “extender” to catalyze further DNA synthesis past the lesion, Dpo2 bridges the activity gap between Dpo4 and Dpo1 in the archaeal TLS process, thus achieving more efficient lesion bypass.

Cisplatin (CDDP)-based chemotherapy is the standard of care in patients with muscle-invasive bladder cancer. However, in a large number of cases, the disease becomes resistant or does not respond to CDDP, and thus progresses and disseminates. In such cases, prognosis of patients is very poor. CDDP manifests its cytotoxic effects mainly through DNA damage induction. Hence, response to CDDP is mainly dependent on DNA damage repair and tolerance mechanisms. Herein, we have examined CDDP response in a panel of the urothelial carcinoma cell (UCC) lines. We characterized these cell lines with regard to viability after CDDP treatment, as well as kinetics of induction and repair of CDDP-induced DNA damage. We demonstrate that repair of CDDP-induced DNA lesions correlates, at least to some extent, with CDDP sensitivity. Furthermore, we monitored expression of the key genes involved in selected DNA repair and tolerance mechanisms, nucleotide excision repair, homologous recombination and translesion DNA synthesis, and show that it differs in the UCC lines and positively correlates with CDDP resistance. Our data indicate that CDDP response in the UCC lines is dependent on DNA damage repair and tolerance factors, which may, therefore, represent valuable therapeutic targets in this malignancy.

Aryl hydrocarbons such as 3-nitrobenzanthrone (NBA), 4-aminobiphenyl (ABP), acetylaminofluorene (AAF), benzo(a)pyrene (BaP), and 1-nitropyrene (NP) form bulky DNA adducts when absorbed by mammalian cells. These chemicals are metabolically activated to reactive forms in mammalian cells and preferentially get attached covalently to the N2 or C8 positions of guanine or the N6 position of adenine. The proportion of N2 and C8 guanine adducts in DNA differs among chemicals. Although these adducts block DNA replication, cells have a mechanism allowing to continue replication by bypassing these adducts: translesion DNA synthesis (TLS). TLS is performed by translesion DNA polymerases—Pol η, κ, ι, and ζ and Rev1—in an error-free or error-prone manner. Regarding the NBA adducts, namely, 2-(2′-deoxyguanosin-N2-yl)-3-aminobenzanthrone (dG-N2-ABA) and N-(2′-deoxyguanosin-8-yl)-3-aminobenzanthrone (dG-C8-ABA), dG-N2-ABA is produced more often than dG-C8-ABA, whereas dG-C8-ABA blocks DNA replication more strongly than dG-N2-ABA. dG-N2-ABA allows for a less error-prone bypass than dG-C8-ABA does. Pol η and κ are stronger contributors to TLS over dG-C8-ABA, and Pol κ bypasses dG-C8-ABA in an error-prone manner. TLS efficiency and error-proneness are affected by the sequences surrounding the adduct, as demonstrated in our previous study on an ABP adduct, N-(2′-deoxyguanosine-8-yl)-4-aminobiphenyl (dG-C8-ABP). Elucidation of the general mechanisms determining efficiency, error-proneness, and the polymerases involved in TLS over various adducts is the next step in the research on TLS. These TLS studies will clarify the mechanisms underlying aryl hydrocarbon mutagenesis and carcinogenesis in more detail.

DNA polymerase (pol) kappa is a Y-family translesion DNA polymerase conserved throughout all domains of life. Pol kappa is special6 ized for the ability to copy DNA containing minor groove DNA adducts, especially N2-dG adducts, as well as to extend primer termini containing DNA damage or mismatched base pairs. Pol kappa generally cannot copy DNA containing major groove modifications or UV-induced photoproducts. Pol kappa can also copy structured or non-B-form DNA, such as microsatellite DNA, common fragile sites, and DNA containing G quadruplexes. Thus, pol kappa has roles both in maintaining and compromising genomic integrity. The expression of pol kappa is altered in several different cancer types, which can lead to genome instability. In addition, many cancer-associated single-nucleotide polymorphisms have been reported in the POLK gene, some of which are associated with poor survival and altered chemotherapy response. Because of this, identifying inhibitors of pol kappa is an active area of research. This review will address these activities of pol kappa, with a focus on lesion bypass and cellular mutagenesis.

Translesion synthesis (TLS) through DNA adducts of antitumor platinum complexes has been an interesting aspect of DNA synthesis in cells treated with these metal-based drugs because of its correlation to drug sensitivity. We utilized model systems employing a DNA lesion derived from a site-specific monofunctional adduct formed by antitumor [PtCl(en)(L)](NO3)2 (complex AMD, en = ethane-1,2-diamine, L = N-[2-(acridin-9-ylamino)ethyl]-N-methylpropionamidine) at a unique G residue. The catalytic efficiency of TLS DNA polymerases, which differ in their processivity and fidelity for the insertion of correct dCTP, with respect to the other incorrect nucleotides, opposite the adduct of AMD, was investigated. For a deeper understanding of the factors that control the bypass of the site-specific adducts of AMD catalyzed by DNA polymerases, we also used microscale thermophoresis (MST) to measure the thermodynamic changes associated with TLS across a single, site-specific adduct formed in DNA by AMD. The relative catalytic efficiency of the investigated DNA polymerases for the insertion of correct dCTP, with respect to the other incorrect nucleotides, opposite the AMD adduct, was reduced. Nevertheless, incorporation of the correct C opposite the G modified by AMD of the template strand was promoted by an increasing thermodynamic stability of the resulting duplex. The reduced relative efficiency of the investigated DNA polymerases may be a consequence of the DNA intercalation of the acridine moiety of AMD and the size of the adduct. The products of the bypass of this monofunctional lesion produced by AMD and DNA polymerases also resulted from the misincorporation of dNTPs opposite the platinated G residues. The MST analysis suggested that thermodynamic factors may contribute to the forces that governed enhanced incorporation of the incorrect dNTPs by DNA polymerases.

High fidelity replicative DNA polymerases are unable to synthesize past DNA adducts that result from diverse chemicals, reactive oxygen species or UV light. To bypass these replication blocks, cells utilize specialized translesion DNA polymerases that are intrinsically error prone and associated with mutagenesis, drug resistance, and cancer. How untimely access of translesion polymerases to DNA is prevented is poorly understood. Here we use co-localization single-molecule spectroscopy (CoSMoS) to follow the exchange of the E. coli replicative DNA polymerase Pol IIIcore with the translesion polymerases Pol II and Pol IV. We find that in contrast to the toolbelt model, the replicative and translesion polymerases do not form a stable complex on one clamp but alternate their binding. Furthermore, while the loading of clamp and Pol IIIcore is highly organized, the exchange with the translesion polymerases is stochastic and is not determined by lesion-recognition but instead a concentration-dependent competition between the polymerases.

DNA damage tolerance (DDT) is a cell function to avoid replication arrest by DNA damage during DNA replication. DDT includes two pathways, translesion DNA synthesis (TLS) and template-switched DNA synthesis (TS). DDT is regulated by ubiquitination of proliferating cell nuclear antigen that binds to double-stranded DNA and functions as scaffold protein for DNA metabolism. TLS is transient DNA synthesis using damaged DNA as a template by error-prone DNA polymerases termed TLS polymerases specialized for DNA damage. TS, in which one newly synthesized strand is utilized as an undamaged template for replication by replicative polymerases, is error-free process. Thus, DDT is not inherently a repair pathway. DDT is a mechanism to tolerate DNA damage, giving priority to DNA synthesis and enabling finish of DNA replication for cell survival and genome stability. DDT is associated with cancer development and thus is of great interest in drug discovery for cancer therapy. This review article describes recent progress in structural studies on protein-protein and protein-DNA complexes involved in TLS and TS, providing the molecular mechanisms of interactions in DDT.

// Xuefei Zhu 1, * , Xiaolu Ma 2, * , Yingfeng Tu 1, * , Min Huang 2 , Hongmei Liu 1 , Fengli Wang 1 , Juanjuan Gong 1 , Jiuqiang Wang 1 , Xiaoling Li 1 , Qian Chen 1 , Hongyan Shen 2 , Shu Zhu 1 , Yun Wang 1 , Yang Liu 2 , Caixia Guo 2 , Tie-Shan Tang 1 1 State Key Laboratory of Membrane Biology, Institute of Zoology, University of Chinese Academy of Sciences, Chinese Academy of Sciences, Beijing 100101, China 2 CAS Key Laboratory of Genomics and Precision Medicine, Beijing Institute of Genomics, University of Chinese Academy of Sciences, Chinese Academy of Sciences, Beijing 100101, China * These authors contributed equally to this work Correspondence to: Caixia Guo, email: guocx@big.ac.cn Tie-Shan Tang, email: tangtsh@ioz.ac.cn Keywords: Parkin, translesion DNA synthesis, ultraviolet radiation, melanoma, Parkinson’s disease Received: January 23, 2017 Accepted: March 27, 2017 Published: April 05, 2017 ABSTRACT Deficiency of Parkin is a major cause of early-onset Parkinson’s disease (PD). Notably, PD patients also exhibit a significantly higher risk in melanoma and other skin tumors, while the mechanism remains largely unknown. In this study, we show that depletion of Parkin causes compromised cell viability and genome stability after ultraviolet (UV) radiation. We demonstrate that Parkin promotes efficient Rad18-dependent proliferating cell nuclear antigen (PCNA) monoubiquitination by facilitating the formation of Replication protein A (RPA)-coated ssDNA upon UV radiation. Furthermore, Parkin is found to physically interact with NBS1 (Nijmegen breakage syndrome 1), and to be required for optimal recruitment of NBS1 and DNA polymerase eta (Polη) to UV-induced damage sites. Consequently, depletion of Parkin leads to increased UV-induced mutagenesis. These findings unveil an important role of Parkin in protecting genome stability through positively regulating translesion DNA synthesis (TLS) upon UV damage, providing a novel mechanistic link between Parkin deficiency and predisposition to skin cancers in PD patients.

Oxidative stress in cells can lead to the accumulation of reactive oxygen species and oxidation of DNA precursors. Oxidized nucleotides such as 2&rsquo, deoxyribo-5-hydroxyuridin (HdU) and 2&rsquo, deoxyribo-5-hydroxymethyluridin (HMdU) can be inserted into DNA during replication and repair. HdU and HMdU have attracted particular interest because they have different effects on damaged-DNA processing enzymes that control the downstream effects of the lesions. Herein, we studied the chemically simulated translesion DNA synthesis (TLS) across the lesions formed by HdU or HMdU using microscale thermophoresis (MST). The thermodynamic changes associated with replication across HdU or HMdU show that the HdU paired with the mismatched deoxyribonucleoside triphosphates disturbs DNA duplexes considerably less than thymidine (dT) or HMdU. Moreover, we also demonstrate that TLS by DNA polymerases across the lesion derived from HdU was markedly less extensive and potentially more mutagenic than that across the lesion formed by HMdU. Thus, DNA polymerization by DNA polymerase &eta, (pol&eta, ), the exonuclease-deficient Klenow fragment of DNA polymerase I (KF&ndash, ), and reverse transcriptase from human immunodeficiency virus type 1 (HIV-1 RT) across these pyrimidine lesions correlated with the different stabilization effects of the HdU and HMdU in DNA duplexes revealed by MST. The equilibrium thermodynamic data obtained by MST can explain the influence of the thermodynamic alterations on the ability of DNA polymerases to bypass lesions induced by oxidative products of pyrimidines. The results also highlighted the usefulness of MST in evaluating the impact of oxidative products of pyrimidines on the processing of these lesions by damaged DNA processing enzymes.

DNA Polymerase Theta (POLQ) is a DNA polymerase involved in error-prone translesion DNA synthesis (TLS) and error-prone repair of DNA double-strand breaks (DSBs). In the present study, we examined whether abnormal POLQ expression may be involved in the pathogenesis of lung adenocarcinoma (LAC). First, we found overexpression of POLQ at both the mRNA and protein levels in LAC, using data from the Cancer Genome Atlas (TCGA) database and by immunohistochemical analysis of our LAC series. POLQ overexpression was associated with an advanced pathologic stage and an increased total number of somatic mutations in LAC. When H1299 human lung cancer cell clones overexpressing POLQ were established and examined, the clones showed resistance to a DSB-inducing chemical in the clonogenic assay and an increased frequency of mutations in the supF forward mutation assay. Further analysis revealed that POLQ overexpression was also positively correlated with Polo Like Kinase 4 (PLK4) overexpression in LAC, and that PLK4 overexpression in the POLQ-overexpressing H1299 cells induced centrosome amplification. Finally, analysis of the TCGA data revealed that POLQ overexpression was associated with an increased somatic mutation load and PLK4 overexpression in diverse human cancers, on the other hand, overexpressions of nine TLS polymerases other than POLQ were associated with an increased somatic mutation load at a much lower frequency. Thus, POLQ overexpression is associated with advanced pathologic stage, increased somatic mutation load, and PLK4 overexpression, the last inducing centrosome amplification, in LAC, suggesting that POLQ overexpression is involved in the pathogenesis of LAC.

7,8-Dihydro-8-oxoguanine (oxoG) is the most abundant oxidative DNA lesion with dual coding properties. It forms both Watson−Crick (anti)oxoG:(anti)C and Hoogsteen (syn)oxoG:(anti)A base pairs without a significant distortion of a B-DNA helix. DNA polymerases bypass oxoG but the accuracy of nucleotide incorporation opposite the lesion varies depending on the polymerase-specific interactions with the templating oxoG and incoming nucleotides. High-fidelity replicative DNA polymerases read oxoG as a cognate base for A while treating oxoG:C as a mismatch. The mutagenic effects of oxoG in the cell are alleviated by specific systems for DNA repair and nucleotide pool sanitization, preventing mutagenesis from both direct DNA oxidation and oxodGMP incorporation. DNA translesion synthesis could provide an additional protective mechanism against oxoG mutagenesis in cells. Several human DNA polymerases of the X- and Y-families efficiently and accurately incorporate nucleotides opposite oxoG. In this review, we address the mutagenic potential of oxoG in cells and discuss the structural basis for oxoG bypass by different DNA polymerases and the mechanisms of the recognition of oxoG by DNA glycosylases and dNTP hydrolases.

Human exonuclease 1 (EXO1), a 5′→3′ exonuclease, contributes to the regulation of the cell cycle checkpoints, replication fork maintenance, and post replicative DNA repair pathways. These processes are required for the resolution of stalled or blocked DNA replication that can lead to replication stress and potential collapse of the replication fork. Failure to restart the DNA replication process can result in double-strand breaks, cell-cycle arrest, cell death, or cellular transformation. In this review, we summarize the involvement of EXO1 in the replication, DNA repair pathways, cell cycle checkpoints, and the link between EXO1 and cancer.

Translesion DNA synthesis (TLS) during S-phase uses specialized TLS DNA polymerases to replicate a DNA lesion, allowing stringent DNA synthesis to resume beyond the offending damage. Human TLS involves the conjugation of ubiquitin to PCNA clamps encircling damaged DNA and the role of this post-translational modification is under scrutiny. A widely-accepted model purports that ubiquitinated PCNA recruits TLS polymerases such as pol η to sites of DNA damage where they may also displace a blocked replicative polymerase. We provide extensive quantitative evidence that the binding of pol η to PCNA and the ensuing TLS are both independent of PCNA ubiquitination. Rather, the unique properties of pols η and δ are attuned to promote an efficient and passive exchange of polymerases during TLS on the lagging strand. DOI: http://dx.doi.org/10.7554/eLife.19788.001

DNA polymerases are essential for genome replication, DNA repair and translesion DNA synthesis (TLS). Broadly, these enzymes belong to two groups: replicative and non-replicative DNA polymerases. A considerable body of data suggests that both groups of DNA polymerases are associated with cancer. Many mutations in cancer cells are either the result of error-prone DNA synthesis by non-replicative polymerases, or the inability of replicative DNA polymerases to proofread mismatched nucleotides due to mutations in 3'-5' exonuclease activity. Moreover, non-replicative, TLS-capable DNA polymerases can negatively impact cancer treatment by synthesizing DNA past lesions generated from treatments such as cisplatin, oxaliplatin, etoposide, bleomycin, and radiotherapy. Hence, the inhibition of DNA polymerases in tumor cells has the potential to enhance treatment outcomes. Here, we review the association of DNA polymerases in cancer from the A and B families, which participate in lesion bypass, and conduct gene replication. We also discuss possible therapeutic interventions that could be used to maneuver the role of these enzymes in tumorigenesis.

The contribution of error-prone DNA polymerases to the DNA damage response has been a subject of great interest in the last decade. Error-prone polymerases are required for translesion DNA synthesis (TLS), a process that involves synthesis past a DNA lesion. Under certain circumstances, TLS polymerases can achieve bypass with good efficiency and fidelity. However, they can also in some cases be mutagenic, and so negative regulators of TLS polymerases would have the important function of inhibiting their recruitment to undamaged DNA templates. Recent work from Livneh's and our groups have provided evidence regarding the role of the cyclin kinase inhibitor p21 as a negative regulator of TLS. Interestingly, both the cyclin dependent kinase (CDK) and proliferating cell nuclear antigen (PCNA) binding domains of p21 are involved in different aspects of the modulation of TLS, affecting both the interaction between PCNA and the TLS-specific pol eta as well as PCNA ubiquitination status. In line with this, p21 was shown to reduce the efficiency but increase the accuracy of TLS. Hence, in absence of DNA damage p21 may work to impede accidental loading of pol eta to undamaged DNA and avoid consequential mutagenesis. After UV irradiation, when TLS plays a decisive role, p21 is progressively degraded. This might allow gradual release of replication fork blockage by TLS polymerases. For these reasons, in higher eukaryotes p21 might represent a key regulator of the equilibrium between mutagenesis and cell survival. Fil: Prives, Carol. Columbia University; Estados Unidos Fil: Gottifredi, Vanesa. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; Argentina

DNA polymerase η (pol η) is best characterized for its ability to perform accurate and efficient translesion DNA synthesis (TLS) through cyclobutane pyrimidine dimers (CPDs). To ensure accurate bypass the polymerase is not only required to select the correct base, but also discriminate between NTPs and dNTPs. Most DNA polymerases have a conserved “steric gate” residue which functions to prevent incorporation of NMPs during DNA synthesis. Here, we demonstrate that the Phe35 residue of Saccharomyces cerevisiae pol η functions as a steric gate to limit the use of ribonucleotides during polymerization both in vitro and in vivo. Unlike the related pol ι enzyme, wild-type pol η does not readily incorporate NMPs in vitro. In contrast, a pol η F35A mutant incorporates NMPs on both damaged and undamaged DNA in vitro with a high degree of base selectivity. An S.cerevisiae strain expressing pol η F35A (rad30-F35A) that is also deficient for nucleotide excision repair (rad1Δ) and the TLS polymerase, pol ζ (rev3Δ), is extremely sensitive to UV-light. The sensitivity is due, in part, to RNase H2 activity, as an isogenic rnh201Δ strain is roughly 50-fold more UV-resistant than its RNH201+ counterpart. Interestingly the rad1Δ rev3Δ rad30-F35A rnh201Δ strain exhibits a significant increase in the extent of spontaneous mutagenesis with a spectrum dominated by 1bp deletions at runs of template Ts. We hypothesize that the increased mutagenesis is due to rA incorporation at these sites and that the short poly rA tract is subsequently repaired in an error-prone manner by a novel repair pathway that is specifically targeted to polyribonucleotide tracks. These data indicate that under certain conditions, pol η can compete with the cell’s replicases and gain access to undamaged genomic DNA. Such observations are consistent with a role for pol η in replicating common fragile sites (CFS) in human cells.

Ultraviolet (UV)-induced DNA damage are removed by nucleotide excision repair (NER) or can be tolerated by specialized translesion synthesis (TLS) polymerases, such as Polη. TLS may act at stalled replication forks or through an S-phase independent gap-filling mechanism. After UVC irradiation, Polη-deficient (XP-V) human cells were arrested in early S-phase and exhibited both single-strand DNA (ssDNA) and prolonged replication fork stalling, as detected by DNA fiber assay. In contrast, NER deficiency in XP-C cells caused no apparent defect in S-phase progression despite the accumulation of ssDNA and a G2-phase arrest. These data indicate that while Polη is essential for DNA synthesis at ongoing damaged replication forks, NER deficiency might unmask the involvement of tolerance pathway through a gap-filling mechanism. ATR knock down by siRNA or caffeine addition provoked increased cell death in both XP-V and XP-C cells exposed to low-dose of UVC, underscoring the involvement of ATR/Chk1 pathway in both DNA damage tolerance mechanisms. We generated a unique human cell line deficient in XPC and Polη proteins, which exhibited both S- and G2-phase arrest after UVC irradiation, consistent with both single deficiencies. In these XP-C/Polη(KD) cells, UVC-induced replicative intermediates may collapse into double-strand breaks, leading to cell death. In conclusion, both TLS at stalled replication forks and gap-filling are active mechanisms for the tolerance of UVC-induced DNA damage in human cells and the preference for one or another pathway depends on the cellular genotype. Fil: Quinet, Annabel. Universidade de Sao Paulo; Brasil. Universite Paris Sud; Francia Fil: Vessoni, Alexandre T. Universidade de Sao Paulo; Brasil Fil: Rocha, Clarissa R. Universidade de Sao Paulo; Brasil Fil: Gottifredi, Vanesa. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquimicas de Buenos Aires; Argentina. Fundación Instituto Leloir; Argentina Fil: Biard, Denis. Commissariat à l’énergie atomique et aux énergies alternatives - CEA ; Francia Fil: Sarasin, Alain. Universite de Paris; Francia Fil: Menck, Carlos F. Universidade de Sao Paulo; Brasil Fil: Stary, Anne. Universite de Paris; Francia

The 15 known Fanconi anemia proteins cooperate in a pathway that regulates DNA interstrand cross-link repair. Recent studies indicate that the Fanconi anemia pathway also controls Rev1-mediated translesion DNA synthesis (TLS). We identified Fanconi anemia-associated protein (FAAP20), an integral subunit of the multisubunit Fanconi anemia core complex. FAAP20 binds to FANCA subunit and is required for stability of the complex and monoubiquitination of FANCD2. FAAP20 contains a ubiquitin-binding zinc finger 4 domain and binds to the monoubiquitinated form of Rev1. FAAP20 binding stabilizes Rev1 nuclear foci and promotes interaction of the Fanconi anemia core with PCNA-Rev1 DNA damage bypass complexes. FAAP20 therefore provides a critical link between the Fanconi anemia pathway and TLS polymerase activity. We propose that the Fanconi anemia core complex regulates cross-link repair by channeling lesions to damage bypass pathways and preventing large DNA insertions and deletions.

Y-family DNA polymerases bypass Pt-GG, the cisplatinDNA double-base lesion, contributing to the cisplatin resistance in tumour cells. To reveal the mechanism, we determined three structures of the Y-family DNA polymerase, Dpo4, in complex with Pt-GG DNA. The crystallographic snapshots show three stages of lesion bypass: the nucleotide insertions opposite the 3 0 G (first insertion) and 5 0 G (second insertion) of Pt-GG, and the primer extension beyond the lesion site. We observed a dynamic process, in which the lesion was converted from an open and angular conformation at the first insertion to a depressed and nearly parallel conformation at the subsequent reaction stages to fit into the active site of Dpo4. The DNA translocation-coupled conformational change may account for additional inhibition on the second insertion reaction. The structures illustrate that Pt-GG disturbs the replicating base pair in the active site, which reduces the catalytic efficiency and fidelity. The in vivo relevance of Dpo4-mediated Pt-GG bypass was addressed by a dpo-4 knockout strain of Sulfolobus solfataricus, which exhibits enhanced sensitivity to cisplatin and proteomic alterations consistent with genomic stress.

The domain Archaea is composed of several subdomains, and prominent among them are the Crenarchaeota and the Euryarchaeota. Biochemically characterized archaeal family Y DNA polymerases (Pols) or DinB homologs, to date, are all from crenarchaeal organisms, especially the genus Sulfolobus. Here, we demonstrate that archaeal family Y Pols fall into five clusters based on phylogenetic analysis. MacDinB-1, the homolog from the euryarchaeon Methanosarcina acetivorans that is characterized in this study, belongs to cluster II. Therefore, MacDinB-1 is different from the Sulfolobus DinB proteins, which are members of cluster I. In addition to translesion DNA synthesis activity, MacDinB-1 synthesized unusually long products ( approximately 7.2 kb) in the presence of its cognate proliferating cell nuclear antigen (PCNA). The PCNA-interacting site in MacDinB-1 was identified by mutational analysis in a C-terminally located heptapeptide akin to a PIP (PCNA-interacting protein) box. In vitro assays from the present report suggested that MacDinB-1 works in an error-free mode to repair cyclobutane pyrimidine dimers. This study on a euryarchaeal DinB homolog provides important insights into the functional diversity of the family Y Pols, and the availability of a genetic system for this archaeon should allow subsequent elucidation of the physiological significance of this enzyme in M. acetivorans cells.