Role In Translesion Synthesis Research Articles

REV7 Monomer Is Unable to Participate in Double Strand Break Repair and Translesion Synthesis but Suppresses Mitotic Errors.

Rev7 is a regulatory protein with roles in translesion synthesis (TLS), double strand break (DSB) repair, replication fork protection, and cell cycle regulation. Rev7 forms a homodimer in vitro using its HORMA (Hop, Rev7, Mad2) domain; however, the functional importance of Rev7 dimerization has been incompletely understood. We analyzed the functional properties of cells expressing either wild-type mouse Rev7 or Rev7K44A/R124A/A135D, a mutant that cannot dimerize. The expression of wild-type Rev7, but not the mutant, rescued the sensitivity of Rev7-/- cells to X-rays and several alkylating agents and reversed the olaparib resistance phenotype of Rev7-/- cells. Using a novel fluorescent host-cell reactivation assay, we found that Rev7K44A/R124A/A135D is unable to promote gap-filling TLS opposite an abasic site analog. The Rev7 dimerization interface is also required for shieldin function, as both Rev7-/- cells and Rev7-/- cells expressing Rev7K44A/R124A/A135D exhibit decreased proficiency in rejoining some types of double strand breaks, as well as increased homologous recombination. Interestingly, Rev7K44A/R124A/A135D retains some function in cell cycle regulation, as it maintains an interaction with Ras-related nuclear protein (Ran) and partially rescues the formation of micronuclei. The mutant Rev7 also rescues the G2/M accumulation observed in Rev7-/- cells but does not affect progression through mitosis following nocodazole release. We conclude that while Rev7 dimerization is required for its roles in TLS, DSB repair, and regulation of the anaphase promoting complex, dimerization is at least partially dispensable for promoting mitotic spindle assembly through its interaction with Ran.

DNA polymerase eta protects human cells against DNA damage induced by the tumor chemotherapeutic temozolomide

Human DNA polymerases can bypass DNA lesions performing translesion synthesis (TLS), a mechanism of DNA damage tolerance. Tumor cells use this mechanism to survive lesions caused by specific chemotherapeutic agents, resulting in treatment relapse. Moreover, TLS polymerases are error-prone and, thus, can lead to mutagenesis, increasing the resistance potential of tumor cells. DNA polymerase eta (pol eta) - a key protein from this group - is responsible for protecting against sunlight-induced tumors. Xeroderma Pigmentosum Variant (XP-V) patients are deficient in pol eta activity, which leads to symptoms related to higher sensitivity and increased incidence of skin cancer. Temozolomide (TMZ) is a chemotherapeutic agent used in glioblastoma and melanoma treatment. TMZ damages cells’ genomes, but little is known about the role of TLS in TMZ-induced DNA lesions. This work investigates the effects of TMZ treatment in human XP-V cells, which lack pol eta, and in its complemented counterpart (XP-V comp). Interestingly, TMZ reduces the viability of XP-V cells compared to TLS proficient control cells. Furthermore, XP-V cells treated with TMZ presented increased phosphorylation of H2AX, forming γH2AX, compared to control cells. However, cell cycle assays indicate that XP-V cells treated with TMZ replicate damaged DNA and pass-through S-phase, arresting in the G2/M-phase. DNA fiber assay also fails to show any specific effect of TMZ-induced DNA damage blocking DNA elongation in pol eta deficient cells. These results show that pol eta plays a role in protecting human cells from TMZ-induced DNA damage, but this can be different from its canonical TLS mechanism. The new role opens novel therapeutic possibilities of using pol eta as a target to improve the efficacy of TMZ-based therapies against cancer.

Abstract 1178: Role of error-prone polymerases on glioma cell resistance to Temozolomide

Abstract Translesion DNA polymerases are capable of replicating damaged DNA without removing lesions, performing translesion synthesis (TLS), a mechanism known by DNA damage tolerance. Tumor cells use this mechanism in order to survive lesions caused by chemotherapy and therefore this may be a strategy that those cells use to resist treatments. Moreover, this process is error-prone and can lead to mutagenesis increasing resistance potential of tumor cells. Little is known about the role of TLS in in tumor therapy with Temozolomide (TMZ). This drug is an alkylating agent that damage DNA. Our aim is to investigate how TMZ affects TLS mutated cells, to understand how these polymerases are related to tumor cells resistance to this chemotherapeutic agent. Hence, we firstly treated POLH mutated cells and analyzed cell proliferation by flow cytometry, cell viability by a colorimetric assay (XTT) and for survival by apoptotic markers (such sub-G1) and clonogenic assays. In fact, POLH mutated cells are more sensitive to TMZ, indicating that TLS mechanisms are important to overcome DNA damage. TCGA data was also used to analyze TLS expression in glioblastoma patients. Curiously, in recurrently patients treated with TMZ, POLK expression was significantly increased. Therefore, we constructed U251-MG CRISPR/CAS9 knockout cells, for POLK and POLI TLS DNA polymerases enzymes genes. Gene KO was validated using Sanger sequencing and mutations resulted in stop codon. These POLI and POLK mutated cells had impaired viability after TMZ treatment. Moreover, genotoxic stress, as indicated by phosphorylated gH2AX, were increased in TMZ-treated cells and was higher in mutated cells compared to Wild-type cells. It is also possible to observe cell cycle arrest, mainly in G2 phase instead of S phase, after 48 h in all three mutated cells, which is not observed in control, wild type, cells. To analyze closer the DNA replication, we also performed fiber assay, surprisingly there was no difference in between KO cells treated with TMZ and WT treated cells! In summary, we can infer that TLS polymerases protect tumor cells from TMZ-induced DNA damage and thus play important roles on overcoming resistance to this drug. As perspectives for this work, we intend to observe further how replication is affected in these mutated cells treated with TMZ, since the lack of difference and the non observation of S phase arrest, may indicate that the role of translesion synthesis may not be the main role in this context. Citation Format: Marcela Teatin Latancia, André Uchimura Bastos, Natália C Moreno, Davi Jardim, Clarissa RR Rocha, Giovana Leandro, Carlos Frederico Martins Menck. Role of error-prone polymerases on glioma cell resistance to Temozolomide [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1178.

DNA polymerase λ promotes error-free replication through Watson–Crick impairing N1-methyl-deoxyadenosine adduct in conjunction with DNA polymerase ζ

In a previous study, we showed that replication through the N1-methyl-deoxyadenosine (1-MeA) adduct in human cells is mediated via three different Polι/Polθ, Polη, and Polζ-dependent pathways. Based on biochemical studies with these Pols, in the Polι/Polθ pathway, we inferred a role for Polι in the insertion of a nucleotide (nt) opposite 1-MeA and of Polθ in extension of synthesis from the inserted nt; in the Polη pathway, we inferred that this Pol alone would replicate through 1-MeA; in the Polζ pathway, however, the Pol required for inserting an nt opposite 1-MeA had remained unidentified. In this study, we provide biochemical and genetic evidence for a role for Polλ in inserting the correct nt T opposite 1-MeA, from which Polζ would extend synthesis. The high proficiency of purified Polλ for inserting a T opposite 1-MeA implicates a role for Polλ—which normally uses W-C base pairing for DNA synthesis—in accommodating 1-MeA in a syn confirmation and forming a Hoogsteen base pair with T. The potential of Polλ to replicate through DNA lesions by Hoogsteen base pairing adds another novel aspect to Polλ’s role in translesion synthesis in addition to its role as a scaffolding component of Polζ. We discuss how the action mechanisms of Polλ and Polζ could be restrained to inserting a T opposite 1-MeA and extending synthesis thereafter, respectively.

DNA methylation profiling of a cnidarian-algal symbiosis using nanopore sequencing.

Symbiosis with protists is common among cnidarians such as corals and sea anemones and is associated with homeostatic and phenotypic changes in the host that could have epigenetic underpinnings, such as methylation of CpG dinucleotides. We leveraged the sensitivity to base modifications of nanopore sequencing to probe the effect of symbiosis with the chlorophyte Elliptochloris marina on methylation in the sea anemone Anthopleura elegantissima. We first validated the approach by comparison of nanopore-derived methylation levels with CpG depletion analysis of a published transcriptome, finding that high methylation levels are associated with CpG depletion as expected. Next, using reads generated exclusively from aposymbiotic anemones, a largely complete draft genome comprising 243 Mb was assembled. Reads from aposymbiotic and symbiotic sea anemones were then mapped to this genome and assessed for methylation using the program Nanopolish, which detects signal disruptions from base modifications as they pass through the nanopore. Based on assessment of 452,841 CpGs for which there was adequate read coverage (approximately 8% of the CpGs in the genome), symbiosis with E. marina was, surprisingly, associated with only subtle changes in the host methylome. However, we did identify one extended genomic region with consistently higher methylation among symbiotic individuals. The region was associated with a DNA polymerase zeta that is noted for its role in translesion synthesis, which opens interesting questions about the biology of this symbiosis. Our study highlights the power and relative simplicity of nanopore sequencing for studies of nucleic acid base modifications in non-model species.

REV7: Jack of many trades.

The HORMA domain protein REV7, also known as MAD2L2, interacts with a variety of proteins and thereby contributes to the establishment of different complexes. With doing so, REV7 impacts a diverse range of cellular processes and gained increasing interest as more of its activities became uncovered. REV7 has important roles in translesion synthesis and mitotic progression, and acts as a central component in the recently discovered shieldin complex that operates in DNA double-strand break repair. Here we discuss the roles of REV7 in its various complexes, focusing on its activity in genome integrity maintenance. Moreover, we will describe current insights on REV7 structural features that allow it to be such a versatile protein.

A novel role of DNA polymerase λ in translesion synthesis in conjunction with DNA polymerase ζ.

By extending synthesis opposite from a diverse array of DNA lesions, DNA polymerase (Pol) ζ performs a crucial role in translesion synthesis (TLS). In yeast and cancer cells, Rev1 functions as an indispensable scaffolding component of Polζ and it imposes highly error-prone TLS upon Polζ. However, for TLS that occurs during replication in normal human cells, Rev1 functions instead as a scaffolding component of Pols η, ι, and κ and Rev1-dependent TLS by these Pols operates in a predominantly error-free manner. The lack of Rev1 requirement for Polζ function in TLS in normal cells suggested that some other protein substitutes for this Rev1 role. Here, we identify a novel role of Polλ as an indispensable scaffolding component of Polζ. TLS studies opposite a number of DNA lesions support the conclusion that as an integral component, Polλ adapts Polζ-dependent TLS to operate in a predominantly error-free manner in human cells, essential for genome integrity and cellular homeostasis.

Abstract 4089: Investigating translesion synthesis DNA polymerases roles on tumor protection after temozolomide induced DNA damage

Abstract Translesion DNA polymerases are capable of replicating damaged DNA, without removing the lesions, performing translesion synthesis (TLS), a mechanism of DNA damage tolerance. Tumor cells use this mechanism in order to survive lesions caused by chemotherapy and, therefore, this may be a strategy that tumor cells use to resist treatments. Moreover, it is an error-prone process and can lead to mutagenesis increasing resistance potential of tumor cells. Little is known about the role of TLS in Temozolomide (TMZ) tumor treatment. Our aim is to investigate how TMZ affects TLS mutated cells. Hence, we firstly treated primary fibroblasts cells lacking TLS mechanisms (XP-V) and analyzed cell proliferation by flow cytometry, cell viability by a colorimetric assay (XTT), for survival by apoptotic markers (such sub-G1) and clonogenic assays. In fact, XP-V cells are more sensitive to TMZ, indicating that TLS mechanisms are important to overcome DNA damage. TCGA data was also used to analyze TLS expression in glioblastoma patients. Curiously, in recurrently patients treated with TMZ, POLK expression was significantly increased. CRISPR/CAS9 knockout (KO) cells of glioblastoma cell lines, such as U251, were constructed for other TLS DNA polymerases enzymes genes, such as Pol Iota and Pol kappa. Gene KO was validated using Sanger sequencing and mutations resulted in stop codon. These pol iota and pol kappa KO cells had impaired viability after TMZ treatment. Moreover, genotoxic stress, as indicated by phosphorylated gH2AX, were increased in TMZ-treated cells. In summary, we can infer that TLS polymerases protect tumor cells from TMZ-induced DNA damage and thus, play important roles on overcoming resistance to this drug. As perspectives for this work, we intend to observe how replication is affected in these KO cells treated with TMZ by DNA Fiber Assays and measure DNA strand breaks in those cells by comet assay. Financial support: FAPESP, CNPq and CAPES. Citation Format: Marcela Teatin Latancia, André Uchimura Bastos, Natália Cestari Moreno, Davi Jardim, Clarissa RR Rocha, Carlos FM Menck. Investigating translesion synthesis DNA polymerases roles on tumor protection after temozolomide induced DNA damage [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4089.

Tumor-Specific Nascent Nine-Peptide-Epitopes Prediction and Bioinformatics Characterization in Human Colorectal Cancer

Background/Aims: Neoantigens are peptides produced by translation of mutant exons and existed in tumor tissues instead of normal tissues, thus, we desired to investigate the antigenic peptide epitopes of cancerspecific neoantigen, to detect the affinity of the nonapeptide with the corresponding Human Leukocyte Antigen I (HLA I) allele molecule, in order to understand the relationship of mutant exon genes of Colorectal Cancer (CRC) patients and the drive genes that are currently known for tumorigenesis of CRC. Methods: The next generation sequencing (NGS) method was used to detect the whole genome sequence and HLA I allele types of tumor tissues and adjacent tissues of 5 CRC patients. pVAC-Seq was applied to identify the nascent nonapeptides generated from exon mutations. The affinity of polypeptides with respective HLA I molecules in CRC was calculated by using NETMHC 4.0 Server. The molecular localization, molecular function, and signal pathways of mutant genes in 5 CRC patients were performed by FunRich 3.1.3 software. The TIMER website was applied to predict the analysis of intratumoral immune cell infiltration associated with the gene mutations in 5 CRC patients. Results: Fifty-six tumor-specific neo-nonapeptides were predicted from 54 different exon gene mutations. The 56 tumor new nonapeptide sequences were different from the shared motif of the HLA I allele. We explored that the tumor-specific nascent nonapeptide mainly bound to HLA-A.02*03, HLA-B.58*01 and HLA-B.11*01, and the affinity analysis results suggested that 14 of the nonapeptides had strong binding force, 20 nonapeptides had weak binding force, and 22 nonapeptides had no binding force. 54 mutant exons of 5 CRC patients were chiefly located in Leading edge membrane, Fanconi anaemia nuclear complex, and Azurophil granule. The molecular functions of these genes were involved in DNA-directed DNA polymerase activity, Vitamin or cofactor transporter activity, and Receptor signaling protein tyrosine kinase activity. 54 gene mutations had key roles in Translesion synthesis by Pol zeta, Translesion synthesis by DNA polymerases bypassing lesion on DNA template, and DNA Damage Bypass. We found that the mutant FMN2 had more infiltration of CD8+ T cells in the tumor than the wild type, and the mutant ZNF717 had more infiltration of CD8+ T cells and neutrophils in the tumor than the wild type, the difference was statistically significant. Conclusion: This study provides a preliminary result to illustrate that the prediction and bioinformatics feature of tumor-specific new nine-peptide-epitopes in CRC. It is hoped that the cancer-specific neoantigen will be used for adjuvant immunotherapy after radical surgery of colorectal cancer, and the mutant genes of CRC can also be used as landmarks for postoperative recurrence and metastasis of colorectal cancer.

Rev7 dimerization is important for assembly and function of the Rev1/Polζ translesion synthesis complex

The translesion synthesis (TLS) polymerases Polζ and Rev1 form a complex that enables replication of damaged DNA. The Rev7 subunit of Polζ, which is a multifaceted HORMA (Hop1, Rev7, Mad2) protein with roles in TLS, DNA repair, and cell-cycle control, facilitates assembly of this complex by binding Rev1 and the catalytic subunit of Polζ, Rev3. Rev7 interacts with Rev3 by a mechanism conserved among HORMA proteins, whereby an open-to-closed transition locks the ligand underneath the "safety belt" loop. Dimerization of HORMA proteins promotes binding and release of this ligand, as exemplified by the Rev7 homolog, Mad2. Here, we investigate the dimerization of Rev7 when bound to the two Rev7-binding motifs (RBMs) in Rev3 by combining in vitro analyses of Rev7 structure and interactions with a functional assay in a Rev7-/- cell line. We demonstrate that Rev7 uses the conventional HORMA dimerization interface both to form a homodimer when tethered by the two RBMs in Rev3 and to heterodimerize with other HORMA domains, Mad2 and p31comet Structurally, the Rev7 dimer can bind only one copy of Rev1, revealing an unexpected Rev1/Polζ architecture. In cells, mutation of the Rev7 dimer interface increases sensitivity to DNA damage. These results provide insights into the structure of the Rev1/Polζ TLS assembly and highlight the function of Rev7 homo- and heterodimerization.

The structure and duplex context of DNA interstrand crosslinks affects the activity of DNA polymerase η.

Several important anti-tumor agents form DNA interstrand crosslinks (ICLs), but their clinical efficiency is counteracted by multiple complex DNA repair pathways. All of these pathways require unhooking of the ICL from one strand of a DNA duplex by nucleases, followed by bypass of the unhooked ICL by translesion synthesis (TLS) polymerases. The structures of the unhooked ICLs remain unknown, yet the position of incisions and processing of the unhooked ICLs significantly influence the efficiency and fidelity of bypass by TLS polymerases. We have synthesized a panel of model unhooked nitrogen mustard ICLs to systematically investigate how the state of an unhooked ICL affects pol η activity. We find that duplex distortion induced by a crosslink plays a crucial role in translesion synthesis, and length of the duplex surrounding an unhooked ICL critically affects polymerase efficiency. We report the synthesis of a putative ICL repair intermediate that mimics the complete processing of an unhooked ICL to a single crosslinked nucleotide, and find that it provides only a minimal obstacle for DNA polymerases. Our results raise the possibility that, depending on the structure and extent of processing of an ICL, its bypass may not absolutely require TLS polymerases.

Aberrant C-terminal domain of polymerase η targets the functional enzyme to the proteosomal degradation pathway.

Xeroderma pigmentosum variant (XP-V) is a rare genetic disease, characterized by sunlight sensitivity and predisposition to cutaneous malignancies. XP-V is caused by a deficiency in DNA polymerase eta (Polη) that plays a pivotal role in translesion synthesis by bypassing UV-induced pyrimidine dimers. Previously we identified a new Polη variant containing two missense mutations, one mutation within the bipartite NLS (T692A) and a second mutation on the stop codon (X714W) leading to a longer protein with an extra 8 amino acids (721 instead of 713 AA). First biochemical analysis revealed that this Polη missense variant was barely detectable by western blot. As this mutant is extremely unstable and is nearly undetectable, a definitive measure of its functional deficit in cells has not been explored. Here we report the molecular and cellular characterization of this missense variant. In cell free extracts, the extra 8 amino acids in the C-terminal of Polη(721) only slightly reduce the bypass efficiency through CPD lesions. In vivo, Polη(721) accumulates in replication factories and interacts with mUb-PCNA albeit at lower level than Polη(wt). XP-V cells overexpressing Polη(721) were only slightly UV-sensitive. Altogether, our data strongly suggest that Polη(721) is functional and that the patient displays a XP-V phenotype because the mutant protein is excessively unstable. We then investigated the molecular mechanisms involved in this excessive proteolysis. We showed that Polη(721) is degraded by the proteasome in an ubiquitin-dependent manner and that this proteolysis is independent of the E3 ligases, CRL4(cdt2) and Pirh2, reported to promote Polη degradation. We then demonstrated that the extra 8 amino acids of Polη(721) do not act as a degron but rather induce a conformational change of the Polη C-terminus exposing its bipartite NLS as well as a sequence close to its UBZ to the ubiquitin/proteasome system. Interestingly we showed that the clinically approved proteasome inhibitor, Bortezomib restores the levels of Polη(721) suggesting that this might be a therapeutic approach to preventing tumor development in certain XP-V patients harboring missense mutations.

The POLD3 subunit of DNA polymerase δ can promote translesion synthesis independently of DNA polymerase ζ.

The replicative DNA polymerase Polδ consists of a catalytic subunit POLD1/p125 and three regulatory subunits POLD2/p50, POLD3/p66 and POLD4/p12. The ortholog of POLD3 in Saccharomyces cerevisiae, Pol32, is required for a significant proportion of spontaneous and UV-induced mutagenesis through its additional role in translesion synthesis (TLS) as a subunit of DNA polymerase ζ. Remarkably, chicken DT40 B lymphocytes deficient in POLD3 are viable and able to replicate undamaged genomic DNA with normal kinetics. Like its counterpart in yeast, POLD3 is required for fully effective TLS, its loss resulting in hypersensitivity to a variety of DNA damaging agents, a diminished ability to maintain replication fork progression after UV irradiation and a significant decrease in abasic site-induced mutagenesis in the immunoglobulin loci. However, these defects appear to be largely independent of Polζ, suggesting that POLD3 makes a significant contribution to TLS independently of Polζ in DT40 cells. Indeed, combining polη, polζ and pold3 mutations results in synthetic lethality. Additionally, we show in vitro that POLD3 promotes extension beyond an abasic by the Polδ holoenzyme suggesting that while POLD3 is not required for normal replication, it may help Polδ to complete abasic site bypass independently of canonical TLS polymerases.

Persistently stalled replication forks inhibit nucleotide excision repair in trans by sequestering Replication protein A

Rev3, the catalytic subunit of DNA polymerase ζ, is essential for translesion synthesis of cytotoxic DNA photolesions, whereas the Rev1 protein plays a noncatalytic role in translesion synthesis. Here, we reveal that mammalian Rev3−/− and Rev1−/− cell lines additionally display a nucleotide excision repair (NER) defect, specifically during S phase. This defect is correlated with the normal recruitment but protracted persistence at DNA damage sites of factors involved in an early stage of NER, while repair synthesis is affected. Remarkably, the NER defect becomes apparent only at 2 h post-irradiation indicating that Rev3 affects repair synthesis only indirectly, rather than performing an enzymatic role in NER. We provide evidence that the NER defect is caused by scarceness of Replication protein A (Rpa) available to NER, resulting from its sequestration at stalled replication forks. Also the induction of replicative stress using hydroxyurea precludes the accumulation of Rpa at photolesion sites, both in Rev3−/− and in wild-type cells. These data support a model in which the limited Rpa pool coordinates replicative stress and NER, resulting in increased cytotoxicity of ultraviolet light when replicative stress exceeds a threshold.

The C-terminal domain of human Rev1 contains independent binding sites for DNA polymerase η and Rev7 subunit of polymerase ζ

Human Rev1 is a translesion synthesis (TLS) DNA polymerase involved in bypass replication across sites of DNA damage and postreplicational gap-filling. Rev1 plays an essential structural role in TLS by providing a binding platform for other TLS polymerases that insert nucleotides across DNA lesions (polη, polι, polκ) and extend the distorted primer-terminus (polς). We use NMR spectroscopy to demonstrate that the Rev1 C-terminal domain utilizes independent interaction interfaces to simultaneously bind a fragment of the ’inserter’ polη and Rev7 subunit of the ’extender’ polς, thereby serving as a cassette that may accommodate several polymerases making them instantaneously available for TLS. Structured summary of protein interactionsREV1, REV3 and REV7physically interact by nuclear magnetic resonance (View interaction), molecular sieving (View interaction) and isothermal titration calorimetry (View interaction).REV3 and REV7bind by molecular sieving (View interaction)REV1 and Polη-RIR peptidebind by nuclear magnetic resonance (View interaction)REV1, REV3, REV7 and Polη-RIR peptidephysically interact by nuclear magnetic resonance (View interaction).

Multifaceted Recognition of Vertebrate Rev1 by Translesion Polymerases ζ and κ

Translesion synthesis is a fundamental biological process that enables DNA replication across lesion sites to ensure timely duplication of genetic information at the cost of replication fidelity, and it is implicated in development of cancer drug resistance after chemotherapy. The eukaryotic Y-family polymerase Rev1 is an essential scaffolding protein in translesion synthesis. Its C-terminal domain (CTD), which interacts with translesion polymerase ζ through the Rev7 subunit and with polymerases κ, ι, and η in vertebrates through the Rev1-interacting region (RIR), is absolutely required for function. We report the first solution structures of the mouse Rev1 CTD and its complex with the Pol κ RIR, revealing an atypical four-helix bundle. Using yeast two-hybrid assays, we have identified a Rev7-binding surface centered at the α2-α3 loop and N-terminal half of α3 of the Rev1 CTD. Binding of the mouse Pol κ RIR to the Rev1 CTD induces folding of the disordered RIR peptide into a three-turn α-helix, with the helix stabilized by an N-terminal cap. RIR binding also induces folding of a disordered N-terminal loop of the Rev1 CTD into a β-hairpin that projects over the shallow α1-α2 surface and creates a deep hydrophobic cavity to interact with the essential FF residues juxtaposed on the same side of the RIR helix. Our combined structural and biochemical studies reveal two distinct surfaces of the Rev1 CTD that separately mediate the assembly of extension and insertion translesion polymerase complexes and provide a molecular framework for developing novel cancer therapeutics to inhibit translesion synthesis.

NMR Structure and Dynamics of the C-Terminal Domain from Human Rev1 and Its Complex with Rev1 Interacting Region of DNA Polymerase η

Rev1 is a translesion synthesis (TLS) DNA polymerase essential for DNA damage tolerance in eukaryotes. In the process of TLS stalled high-fidelity replicative DNA polymerases are temporarily replaced by specialized TLS enzymes that can bypass sites of DNA damage (lesions), thus allowing replication to continue or postreplicational gaps to be filled. Despite its limited catalytic activity, human Rev1 plays a key role in TLS by serving as a scaffold that provides an access of Y-family TLS polymerases polη, ι, and κ to their cognate DNA lesions and facilitates their subsequent exchange to polζ that extends the distorted DNA primer-template. Rev1 interaction with the other major human TLS polymerases, polη, ι, κ, and the regulatory subunit Rev7 of polζ, is mediated by Rev1 C-terminal domain (Rev1-CT). We used NMR spectroscopy to determine the spatial structure of the Rev1-CT domain (residues 1157-1251) and its complex with Rev1 interacting region (RIR) from polη (residues 524-539). The domain forms a four-helix bundle with a well-structured N-terminal β-hairpin docking against helices 1 and 2, creating a binding pocket for the two conserved Phe residues of the RIR motif that upon binding folds into an α-helix. NMR spin-relaxation and NMR relaxation dispersion measurements suggest that free Rev1-CT and Rev1-CT/polη-RIR complex exhibit μs-ms conformational dynamics encompassing the RIR binding site, which might facilitate selection of the molecular configuration optimal for binding. These results offer new insights into the control of TLS in human cells by providing a structural basis for understanding the recognition of the Rev1-CT by Y-family DNA polymerases.

Temporally distinct translesion synthesis pathways for ultraviolet light-induced photoproducts in the mammalian genome

Replicative polymerases (Pols) arrest at damaged DNA nucleotides, which induces ubiquitination of the DNA sliding clamp PCNA (PCNA-Ub) and DNA damage signaling. PCNA-Ub is associated with the recruitment or activation of translesion synthesis (TLS) DNA polymerases of the Y family that can bypass the lesions, thereby rescuing replication and preventing replication fork collapse and consequent formation of double-strand DNA breaks. Here, we have used gene-targeted mouse embryonic fibroblasts to perform a comprehensive study of the in vivo roles of PCNA-Ub and of the Y family TLS Pols η, ι, κ, Rev1 and the B family TLS Polζ in TLS and in the suppression of DNA damage signaling and genome instability after exposure to UV light. Our data indicate that TLS Pols ι and κ and the N-terminal BRCT domain of Rev1, that previously was implicated in the regulation of TLS, play minor roles in TLS of DNA photoproducts. PCNA-Ub is critical for an early TLS pathway that replicates both strongly helix-distorting (6-4) pyrimidine–pyrimidone ((6-4)PP) and mildly distorting cyclobutane pyrimidine dimer (CPD) photoproducts. The role of Polη is mainly restricted to early TLS of CPD photoproducts, whereas Rev1 and, in particular, Polζ are essential for the bypass of (6-4)PP photoproducts, both early and late after exposure. Thus, structurally distinct photoproducts at the mammalian genome are bypassed by different TLS Pols in temporally different, PCNA-Ub-dependent and independent fashions.

A novel ubiquitin binding mode in the S. cerevisiae translesion synthesis DNA polymerase η

The ubiquitin binding zinc finger (UBZ) domain in the C-terminal portion of Polη has been found to interact with ubiquitin. However, the affinity between the Polη UBZ and ubiquitin was shown to be low with a previously reported K(d) of 73-81 μM. This low-affinity binding between Polη UBZ and ubiquitin has been difficult to reconcile with its presumed role in translesion synthesis as suggested by genetic and cell biology studies. In this work, we constructed a minimal S. cerevisiae Polη UBZ domain and probed the Polη UBZ-ubiquitin interaction using a surface plasmon resonance (SPR) technique. Our quantitative binding data between the wild-type or mutant Polη UBZ and ubiquitin revealed an interesting divergence between the Polη UBZ from S. cerevisiae and humans. Moreover, we found that the C-terminal portion of yeast Polη (amino acid 515-632) binds ubiquitin with a much higher affinity than the minimal UBZ domain. Further, distinct ubiquitin-binding kinetics were observed for the C-terminal portion of Polη and the isolated UBZ domain. This observation raised the interesting possibility that the Polη C-terminal portion binds ubiquitin in a novel mode that affords higher affinity. Our findings have broader implication in understanding the generally weak interaction between the known ubiquitin-binding domains and ubiquitin.

Ubiquitin-dependent regulation of translesion polymerases

In response to DNA damage, TLS (translesion synthesis) allows replicative bypass of various DNA lesions, which stall normal replication. TLS is achieved by low-fidelity polymerases harbouring less stringent active sites. In humans, Y-family polymerases together with Pol zeta (polymerase zeta) are responsible for TLS across different types of damage. Protein-protein interaction contributes significantly to the regulation of TLS. REV1 plays a central role in TLS because it interacts with all other Y-family members and Pol zeta. Ubiquitin-dependent regulatory mechanisms also play important roles in TLS. Ubiquitin-binding domains have been found in TLS polymerases and they might be required for TLS activity. Mono-ubiquitination of PCNA (proliferating-cell nuclear antigen), the central scaffold of TLS polymerases, is thought to promote TLS. In addition, both non-proteolytic and proteolytic polyubiquitination of PCNA and TLS polymerases has been demonstrated. Owing to their low fidelity, the recruitment of TLS polymerases is strictly restricted to stalled replication forks.