Eukaryotic DNA damage tolerance and translesion synthesis through covalent modifications of PCNA

In addition to well-defined DNA repair pathways, all living organisms have evolved mechanisms to avoid cell death caused by replication fork collapse at a site where replication is blocked due to disruptive covalent modifications of DNA. The term DNA damage tolerance （DDT） has been employed loosely to include a collection of mechanisms by which cells survive replication-blocking lesions with or without associated genomic instability. Recent genetic analyses indicate that DDT in eukaryotes, from yeast to human, consists of two parallel pathways with one being error-free and another highly mutagenic. Interestingly, in budding yeast, these two pathways are mediated by sequential modifications of the proliferating cell nuclear antigen （PCNA） by two ubiquitination complexes Rad6-Rad18 and Mms2-Ubc13-Rad5. Damage-induced monoubiquitination of PCNA by Rad6-Rad18 promotes translesion synthesis （TLS） with increased mutagenesis, while subsequent polyubiquitination of PCNA at the same K164 residue by Mms2-Ubc13-Rad5 promotes error-free lesion bypass. Data obtained from recent studies suggest that the above mechanisms are conserved in higher eukaryotes. In particular, mammals contain multiple specialized TLS polymerases. Defects in one of the TLS polymerases have been linked to genomic instability and cancer.     

Ribonucleotide discrimination by translesion synthesis DNA polymerases

The well-being of all living organisms relies on the accurate duplication of their genomes. This is usually achieved by highly elaborate replicase complexes which ensure that this task is accomplished timely and efficiently. However, cells often must resort to the help of various additional “specialized” DNA polymerases that gain access to genomic DNA when replication fork progression is hindered. One such specialized polymerase family consists of the so-called “translesion synthesis” (TLS) polymerases; enzymes that have evolved to replicate damaged DNA. To fulfill their main cellular mission, TLS polymerases often must sacrifice precision when selecting nucleotide substrates. Low base-substitution fidelity is a well-documented inherent property of these enzymes. However, incorrect nucleotide substrates are not only those which do not comply with Watson–Crick base complementarity, but also those whose sugar moiety is incorrect. Does relaxed base-selectivity automatically mean that the TLS polymerases are unable to efficiently discriminate between ribonucleoside triphosphates and deoxyribonucleoside triphosphates that differ by only a single atom? Which strategies do TLS polymerases employ to select suitable nucleotide substrates? In this review, we will collate and summarize data accumulated over the past decade from biochemical and structural studies, which aim to answer these questions.     

Non‐recombinogenic roles for Rad52 in translesion synthesis during DNA damage tolerance

DNA damage tolerance relies on homologous recombination (HR) and translesion synthesis (TLS) mechanisms to fill in the ssDNA gaps generated during passing of the replication fork over DNA lesions in the template. Whereas TLS requires specialized polymerases able to incorporate a dNTP opposite the lesion and is error‐prone, HR uses the sister chromatid and is mostly error‐free. We report that the HR protein Rad52—but not Rad51 and Rad57—acts in concert with the TLS machinery (Rad6/Rad18‐mediated PCNA ubiquitylation and polymerases Rev1/Pol ζ) to repair MMS and UV light‐induced ssDNA gaps through a non‐recombinogenic mechanism, as inferred from the different phenotypes displayed in the absence of Rad52 and Rad54 (essential for MMS‐ and UV‐induced HR); accordingly, Rad52 is required for efficient DNA damage‐induced mutagenesis. In addition, Rad52, Rad51, and Rad57, but not Rad54, facilitate Rad6/Rad18 binding to chromatin and subsequent DNA damage‐induced PCNA ubiquitylation. Therefore, Rad52 facilitates the tolerance process not only by HR but also by TLS through Rad51/Rad57‐dependent and ‐independent processes, providing a novel role for the recombination proteins in maintaining genome integrity.     

Multiple two-polymerase mechanisms in mammalian translesion DNA synthesis

The encounter of replication forks with DNA lesions may lead to fork arrest and/or the formation of single-stranded gaps. A major strategy to cope with these replication irregularities is translesion DNA replication (TLS), in which specialized error-prone DNA polymerases bypass the blocking lesions. Recent studies suggest that TLS across a particular DNA lesion may involve as many as four different TLS polymerases, acting in two-polymerase reactions in which insertion by a particular polymerase is followed by extension by another polymerase. Insertion determines the accuracy and mutagenic specificity of the TLS reaction, and is carried out by one of several polymerases such as polη, polκ or polι. In contrast, extension is carried out primarily by polζ. In cells from XPV patients, which are deficient in TLS across cyclobutane pyrimidine dimers (CPD) due to a deficiency in polη, TLS is carried out by at least two backup reactions each involving two polymerases: One reaction involves polκ and polζ, and the other polι and polζ. These mechanisms may also assist polη in normal cells under an excessive amount of UV lesions.     

Manganese substantially alters the dynamics of translesion DNA synthesis

The effect of metal ion substitution on the dynamics of translesion DNA synthesis catalyzed by the bacteriophage T4 DNA polymerase was quantitatively evaluated through steady-state and transient kinetic techniques. Substitution of Mn(2+) for Mg(2+) enhances the steady-state rate of dNMP misinsertion opposite an abasic site by 11-34-fold. At the molecular level, the enhancement in translesion DNA synthesis reflects a substantial increase in the rate of the conformational change preceding phosphoryl transfer for all dNTPs that were tested. This is best illustrated by the biphasic pre-steady-state time course of dAMP insertion opposite an abasic site which indicates that a step after chemistry is rate-limiting for steady-state enzyme turnover. Furthermore, the k(pol) value of 40 s(-1) measured under single-turnover reaction conditions is 20-fold greater than the k(cat) value of 2 s(-1) measured for steady-state enzyme turnover. Finally, the low elemental effect ( approximately 2.4-fold reduction in k(pol)) measured by substituting the alpha-thiotriphosphate analogue for dATP further argues that chemistry is not rate-limiting. In contrast to the biphasic insertion of dAMP, pre-steady-state time courses for the insertion of dCMP, dGMP, or dTMP opposite an abasic site were linear. Nearly identical k(pol) values ( approximately 1 s(-1)) were measured for the insertion of dCMP, dGMP, and dTMP opposite the abasic site using single-turnover conditions. However, the large elemental effects of 27 and 70 measured by substituting the alpha-thiotriphosphate analogues for dCTP and dGTP, respectively, suggest that phosphoryl transfer may be the rate-limiting step for their insertion opposite the abasic site. Various models are discussed in an attempt to explain the effect of metal ion substitution on the dynamics of translesion DNA replication.     

Error-prone DNA repair and translesion DNA synthesis. II: The inducible SOS hypothesis

Evelyn Witkin hypothesized in 1967 that bacterial cell division is controlled by a repressor which, like the lambda repressor, is inactivated by a complex process that starts with the presence of replication-blocking lesions in the DNA. She further suggested that this might not be the only cellular function to show induction by DNA damage. Three years later, Miroslav Radman, in a privately circulated note, proposed that one such function might be an inaccurate (mutation-prone) DNA polymerase under the control of the recA and lexA genes. Thus was born the SOS hypothesis.     

Enhancing the "A-rule" of translesion DNA synthesis: promutagenic DNA synthesis using modified nucleoside triphosphates

Abasic sites are mutagenic DNA lesions formed as a consequence of inappropriate modifications to the functional groups present on purines and pyrimidines. In this paper we quantify the ability of the high-fidelity bacteriophage T4 DNA polymerase to incorporate various promutagenic alkylated nucleotides opposite and beyond this class of non-instructional DNA lesions. Kinetic analyses reveal that modified nucleotides such as N6-methyl-dATP and O6-methyl-dGTP are incorporated opposite an abasic site far more effectively than their unmodified counterparts. The enhanced incorporation is caused by a 10-fold increase in kpol values that correlates with an increase in hydrophobicity as well as changes in the tautomeric form of the nucleobase to resemble adenine. These biophysical features lead to enhanced base-stacking properties that also contribute toward their ability to be easily extended when paired opposite the non-instructional DNA lesion. Surprisingly, misincorporation opposite templating DNA is not enhanced by the increased base-stacking properties of most modified purines. The dichotomy in promutagenic DNA synthesis catalyzed by a high-fidelity polymerase indicates that the dynamics for misreplicating a miscoding versus a non-instructional DNA lesion are different. The collective data set is used to propose models accounting for synergistic enhancements in mutagenesis and the potential to develop treatment-related malignancies as a consequence of utilizing DNA-damaging agents as chemotherapeutic agents.     

Involvement of vertebrate Polkappa in translesion DNA synthesis across DNA monoalkylation damage

DNA lesions that escape excision repair pathways can cause arrested DNA replication. This replication block can be processed by translesion DNA synthesis (TLS), which is carried out by a number of specialized DNA polymerases. A sequential lesion bypass model has been proposed; one of the lesion-specific polymerases inserts nucleotide(s) opposite the damaged template, followed by extension from the inserted nucleotide by the same or another polymerase. Polzeta and Polkappa have been proposed as candidates for executing the extension step in eukaryotic cells. We previously disrupted separately Rev3, the catalytic subunit of Polzeta, and Polkappa in chicken B lymphocyte DT40 cells. We found that each cell line showed significant UV sensitivity, implying that both contribute to UV radiation damage repair. In the present studies we generated REV3(-/-)POLK(/-) double knock-out cells to determine whether they participate in the same or different pathways. The double mutant was viable and proliferated with the same kinetics as parental REV3(-/-) cells. The cells showed the same sensitivity as REV3(-/-) cells to UV, ionizing radiation, and chemical cross-linking agents. In contrast, they were more sensitive than REV3(-/-) cells to monofunctional alkylating agents, even though POLK(/-) cells barely exhibited increased sensitivity to those. Moreover Polk-deficient mouse embryonic stem and fibroblast cells, both of which have previously been shown to be sensitive to UV radiation, also showed moderate sensitivity to methyl methanesulfonate, a monofunctional alkylating agent. These data imply that Polkappa has a function in TLS past alkylated base adducts as well as UV radiation DNA damage in vertebrates.     

Send in the clamps: control of DNA translesion synthesis in eukaryotes

The replication of damaged DNA templates by translesion synthesis (TLS) is associated with mutagenesis and carcinogenesis. This perspective discusses the different levels at which TLS may be controlled and proposes a model for TLS of severely helix-distorting DNA lesions that includes a decisive role for the Rad9-Hus1-Rad1 DNA-damage-signaling clamp. The dual involvement of this clamp in both DNA-damage signaling and TLS may have profound implications in determining cellular responses to DNA damage.     

The fidelity of DNA synthesis by eukaryotic replicative and translesion synthesis polymerases

In their seminal publication describing the structure of the DNA double helix , Watson and Crick wrote what may be one of the greatest understatements in the scientific literature, namely that ＂It has not escaped our notice that the specific pairing we have postulated immediately suggests a possible copying mechanism for the genetic material.＂ Half a century later, we more fully appreciate what a huge challenge it is to replicate six billion nucleotides with the accuracy needed to stably maintain the human genome over many generations. This challenge is perhaps greater than was realized 50 years ago, because subsequent studies have revealed that the genome can be destabilized not only by environmental stresses that generate a large number and variety of potentially cytotoxic and mutagenic lesions in DNA but also by various sequence motifs of normal DNA that present challenges to replication. Towards a better understanding of the many determinants of genome stability, this chapter reviews the fidelity with which undamaged and damaged DNA is copied, with a focus on the eukaryotic B- and Y-family DNA polymerases, and considers how this fidelity is achieved.     

Error-prone DNA repair and translesion synthesis: focus on the replication fork

Dean Rupp and Paul Howard-Flanders showed that, following exposure to ultraviolet light, bacteria deficient in nucleotide excision repair synthesised DNA with minimal delay and in pieces roughly the size of the distances between pyrimidine dimmers. The discontinuities or gaps between these pieces were subsequently sealed. This led directly to the hypothesis of translesion synthesis.     

Structure-dependent bypass of DNA interstrand crosslinks by translesion synthesis polymerases

DNA interstrand crosslinks (ICLs), inhibit DNA metabolism by covalently linking two strands of DNA and are formed by antitumor agents such as cisplatin and nitrogen mustards. Multiple complex repair pathways of ICLs exist in humans that share translesion synthesis (TLS) past a partially processed ICL as a common step. We have generated site-specific major groove ICLs and studied the ability of Y-family polymerases and Pol ζ to bypass ICLs that induce different degrees of distortion in DNA. Two main factors influenced the efficiency of ICL bypass: the length of the dsDNA flanking the ICL and the length of the crosslink bridging two bases. Our study shows that ICLs can readily be bypassed by TLS polymerases if they are appropriately processed and that the structure of the ICL influences which polymerases are able to read through it.     

PIDD orchestrates translesion DNA synthesis in response to UV irradiation

PIDD has been implicated in survival and apoptotic pathways in response to DNA damage, and a role for PIDD was recently identified in non-homologous end-joining (NHEJ) repair induced by γ-irradiation. Here, we present an interaction of PIDD with PCNA, first identified in a proteomics screen. PCNA has essential functions in DNA replication and repair following UV irradiation. Translesion synthesis (TLS) is a process that prevents UV irradiation-induced replication blockage and is characterized by PCNA monoubiquitination and interaction with the TLS polymerase eta (polη). Both of these processes are inhibited by p21. We report that PIDD modulates p21-PCNA dissociation, and promotes PCNA monoubiquitination and interaction with polη in response to UV irradiation. Furthermore, PIDD deficiency leads to a defect in TLS that is associated, both in vitro and in vivo, with cellular sensitization to UV-induced apoptosis. Thus, PIDD performs key functions upon UV irradiation, including TLS, NHEJ, NF-κB activation and cell death.     

Replication of damaged DNA by translesion synthesis in human cells

Most types of DNA damage block the passage of the replication machinery. In order to bypass these blocks, cells employ special translesion synthesis (TLS) DNA polymerases, which have lower stringency than replicative polymerases. DNA polymerase eta is the major polymerase responsible for bypassing UV lesions in DNA and its absence results in the variant form of the genetic disorder, xeroderma pigmentosum. Other TLS polymerases have specificities for different types of damage, but their precise roles inside the cell have not yet been established. These polymerases are located in replication factories during DNA replication and the polymerase sliding clamp PCNA plays an important role in mediating switching between different polymerases.     

The biochemical requirements of DNA polymerase V-mediated translesion synthesis revisited

In addition to replicative DNA polymerases, cells contain specialized DNA polymerases involved in processes such as lesion tolerance, mutagenesis and immunoglobulin diversity. In Escherichia coli, DNA polymerase V (Pol V), encoded by the umuDC locus, is involved in translesion synthesis (TLS) and mutagenesis. Genetic studies have established that mutagenesis requires both UmuC and a proteolytic product of UmuD (UmuD'). In addition, RecA protein and the replication processivity factor, the beta-clamp, were genetically found to be essential co-factors for mutagenesis. Here, we have reconstituted Pol V-mediated bypass of three common replication-blocking lesions, namely the two major UV-induced lesions and a guanine adduct formed by a chemical carcinogen (G-AAF) under conditions that fulfil these in vivo requirements. Two co-factors are essential for efficient Pol V-mediated lesion bypass: (i) a DNA substrate onto which the beta-clamp is stably loaded; and (ii) an extended single-stranded RecA/ATP filament assembled downstream from the lesion site. For efficient bypass, Pol V needs to interact simultaneously with the beta-clamp and the 3' tip of the RecA filament. Formation of an extended RecA/ATP filament and stable loading of the beta-clamp are best achieved on long single-stranded circular DNA templates. In contrast to previously published data, the single-stranded DNA-binding protein (SSB) is not absolutely required for Pol V-mediated lesion bypass provided ATP, instead of ATPgammaS, activates the RecA filament. Further discrepancies with the existing literature are explainable by the use of either inadequate DNA substrates or a UmuC fusion protein instead of native Pol V.     

Efficiency, fidelity and enzymatic switching during translesion DNA synthesis

More than half of the 16 human DNA polymerases may have some role in DNA replication and potentially modulate the biological effects of DNA template lesions that impede replication fork progression. As one approach to understand how multiple polymerases are coordinated at the fork, we recently quantified the efficiency and fidelity with which one particular translesion synthesis enzyme, human DNA polymerase eta, copies templates containing cis-syn thymine dimers. Several observations from that study were unanticipated. Here we discuss the structural and biological implications of those results in light of earlier studies of translesion synthesis.     

Mechanisms of accurate translesion synthesis by human DNA polymerase eta

The XPV (xeroderma pigmentosum variant) gene encodes human DNA polymerase eta (pol eta), which is involved in the replication of damaged DNA. Pol eta catalyzes efficient and accurate translesion synthesis past cis-syn cyclobutane di-thymine lesions. Here we show that human pol eta can catalyze translesion synthesis past an abasic (AP) site analog, N-2-acetylaminofluorene (AAF)-modified guanine, and a cisplatin-induced intrastrand cross-link between two guanines. Pol eta preferentially incorporated dAMP and dGMP opposite AP, and dCMP opposite AAF-G and cisplatin-GG, but other nucleotides were also incorporated opposite these lesions. However, after incorporating an incorrect nucleotide opposite a lesion, pol eta could not continue chain elongation. In contrast, after incorporating the correct nucleotide opposite a lesion, pol eta could continue chain elongation, whereas pol alpha could not. Thus, the fidelity of translesion synthesis by human pol eta relies not only on the ability of this enzyme to incorporate the correct nucleotide opposite a lesion, but also on its ability to elongate only DNA chains that have a correctly incorporated nucleotide opposite a lesion.     

Dual roles for DNA polymerase eta in homologous DNA recombination and translesion DNA synthesis

Chicken B lymphocyte precursors and DT40 cells diversify their immunoglobulin-variable (IgV) genes through homologous recombination (HR)-mediated Ig gene conversion. To identify DNA polymerases that are involved in Ig gene conversion, we created DT40 clones deficient in DNA polymerase eta (poleta), which, in humans, is defective in the variant form of xeroderma pigmentosum (XP-V). Poleta is an error-prone translesion DNA synthesis polymerase that can bypass UV damage-induced lesions and is involved in IgV hypermutation. Like XP-V cells, poleta-disrupted (poleta) clones exhibited hypersensitivity to UV. Remarkably, poleta cells showed a significant decrease in the frequency of both Ig gene conversion and double-strand break-induced HR when compared to wild-type cells, and these defects were reversed by complementation with human poleta. Our findings identify a DNA polymerase that carries out DNA synthesis for physiological HR and provides evidence that a single DNA polymerase can play multiple cellular roles.