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DNA mismatch repair (MMR) in eukaryotes is believed to occur post-replicatively, wherein nicks or gaps in the nascent DNA strand are suggested to serve as strand discrimination signals. However, how such signals are generated in the nascent leading strand has remained unclear. Here we examine the alternative possibility that MMR occurs in conjunction with the replication fork. To this end, we utilize mutations in the PCNA interacting peptide (PIP) domain of the Pol3 or Pol32 subunit of DNA polymerase δ (Polδ) and show that these pip mutations suppress the greatly elevated mutagenesis in yeast strains harboring the pol3-01 mutation defective in Polδ proofreading activity. And strikingly, they suppress the synthetic lethality of pol3-01 pol2-4 double mutant strains, which arises from the vastly enhanced mutability due to defects in the proofreading functions of both Polδ and Polε. Our finding that suppression of elevated mutagenesis in pol3-01 by the Polδ pip mutations requires intact MMR supports the conclusion that MMR operates at the replication fork in direct competition with other mismatch removal processes and with extension of synthesis from the mispair by Polδ. Furthermore, the evidence that Polδ pip mutations eliminate almost all the mutability of pol2-4 msh2Δ or pol3-01 pol2-4 adds strong support for a major role of Polδ in replication of both the leading and lagging DNA strands.

DNA polymerase ζ (Polζ) belongs to the same B-family as high-fidelity replicative polymerases, yet is specialized for the extension reaction in translesion DNA synthesis (TLS). Despite its importance in TLS, the structure of Polζ is unknown. We present cryo-EM structures of the Saccharomyces cerevisiae Polζ holoenzyme in the act of DNA synthesis (3.1 A) and without DNA (4.1 A). Polζ displays a pentameric ring-like architecture, with catalytic Rev3, accessory Pol31‚ Pol32 and two Rev7 subunits forming an uninterrupted daisy chain of protein–protein interactions. We also uncover the features that impose high fidelity during the nucleotide-incorporation step and those that accommodate mismatches and lesions during the extension reaction. Collectively, we decrypt the molecular underpinnings of Polζ’s role in TLS and provide a framework for new cancer therapeutics. Cryo-EM structures of free and DNA-bound pol ζ holoenzyme from budding yeast reveal how this DNA polymerase ensures fidelity and facilitates lesion bypass during translesion DNA synthesis.

The action mechanisms revealed by the biochemical and structural analyses of replicative and translesion synthesis (TLS) DNA polymerases (Pols) are retained in their cellular roles. In this regard, DNA polymerase θ differs from other Pols in that whereas purified Polθ misincorporates an A opposite 1,N(6)-ethenodeoxyadenosine (ϵdA) using an abasic-like mode, Polθ performs predominantly error-free TLS in human cells. To test the hypothesis that Polθ adopts a different mechanism for replicating through ϵdA in human cells than in the purified Pol, here we analyze the effects of mutations in the two highly conserved tyrosine residues, Tyr-2387 and Tyr-2391, in the Polθ active site. Our findings that these residues are indispensable for TLS by the purified Pol but are not required in human cells, as well as other findings, provide strong evidence that the Polθ active site is reconfigured in human cells to stabilize ϵdA in the syn conformation for Hoogsteen base pairing with the correct nucleotide. The evidence that a DNA polymerase can configure its active site entirely differently in human cells than in the purified Pol establishes a new paradigm for DNA polymerase function.

Eukaryotic cells harbor two DNA-binding clamps, proliferating cell nuclear antigen (PCNA), and another clamp commonly referred to as 9-1-1 clamp. In contrast to the essential role of PCNA in DNA replication as a sliding clamp for DNA polymerase (Pol) δ, no such role in DNA synthesis has been identified for the human 9-1-1 clamp or the orthologous yeast 17-3-1 clamp. The only role identified for either the 9-1-1 or 17-3-1 clamp is in the recruitment of signal transduction kinases, which affect the activation of cell cycle checkpoints in response to DNA damage. However, unlike the loading of PCNA by the replication factor C (RFC) clamp loader onto 3'-recessed DNA junctions for processive DNA synthesis by Polδ, the 17-3-1 clamp or the 9-1-1 clamp is loaded by their respective clamp loader Rad24-RFC or RAD17-RFC onto the 5'-recessed DNA junction of replication protein A-coated DNA for the recruitment of signal transduction kinases. Here, we identify a novel role of 17-3-1 clamp as a sliding clamp for DNA synthesis by Polε. We provide evidence that similar to the loading of PCNA by RFC, the 17-3-1 clamp is loaded by the Rad24-RFC clamp loader at the 3'-recessed DNA junction in an ATP-dependent manner. However, unlike PCNA, the 17-3-1 clamp does not enhance the processivity of DNA synthesis by Polε; instead, it greatly increases the catalytic efficiency of Polε for correct nucleotide incorporation. Furthermore, we show that the same PCNA-interacting peptide domain in the polymerase 2 catalytic subunit mediates Polε interaction with the 17-3-1 clamp and with PCNA.

Cytarabine (AraC) is the mainstay for the treatment of acute myeloid leukemia. Although complete remission is observed in a large proportion of patients, relapse occurs in almost all the cases. The chemotherapeutic action of AraC derives from its ability to inhibit DNA synthesis by the replicative polymerases (Pols); the replicative Pols can insert AraCTP at the 3′ terminus of the nascent DNA strand, but they are blocked at extending synthesis from AraC. By extending synthesis from the 3′-terminal AraC and by replicating through AraC that becomes incorporated into DNA, translesion synthesis (TLS) DNA Pols could reduce the effectiveness of AraC in chemotherapy. Here we identify the TLS Pols required for replicating through the AraC templating residue and determine their error-proneness. We provide evidence that TLS makes a consequential contribution to the replication of AraC-damaged DNA; that TLS through AraC is conducted by three different pathways dependent upon Polη, Polι, and Polν, respectively; and that TLS by all these Pols incurs considerable mutagenesis. The prominent role of TLS in promoting proficient and mutagenic replication through AraC suggests that TLS inhibition in acute myeloid leukemia patients would increase the effectiveness of AraC chemotherapy; and by reducing mutation formation, TLS inhibition may dampen the emergence of drug-resistant tumors and thereby the high incidence of relapse in AraC-treated patients.

Cytarabine (AraC) is the mainstay chemotherapy for acute myeloid leukemia (AML). Whereas initial treatment with AraC is usually successful, most AML patients tend to relapse, and AraC treatment-induced mutagenesis may contribute to the development of chemo-resistant leukemic clones. We show here that whereas the high-fidelity replicative polymerase Polδ is blocked in the replication of AraC, the lower-fidelity translesion DNA synthesis (TLS) polymerase Polη is proficient, inserting both correct and incorrect nucleotides opposite a template AraC base. Furthermore, we present high-resolution crystal structures of human Polη with a template AraC residue positioned opposite correct (G) and incorrect (A) incoming deoxynucleotides. We show that Polη can accommodate local perturbation caused by the AraC via specific hydrogen bonding and maintain a reaction-ready active site alignment for insertion of both correct and incorrect incoming nucleotides. Taken together, the structures provide a novel basis for the ability of Polη to promote AraC induced mutagenesis in relapsed AML patients.

Here we show that translesion synthesis (TLS) opposite 1,N6-ethenodeoxyadenosine (εdA), which disrupts Watson–Crick base pairing, occurs via Polι/Polζ-, Rev1-, and Polθ-dependent pathways. The requirement of Polι/Polζ is consistent with the ability of Polι to incorporate nucleotide opposite εdA by Hoogsteen base pairing and of Polζ to extend synthesis. Rev1 polymerase and Polθ conduct TLS opposite εdA via alternative error-prone pathways. Strikingly, in contrast to extremely error-prone TLS opposite εdA by purified Polθ, it performs predominantly error-free TLS in human cells. Reconfiguration of the active site opposite εdA would provide Polθ the proficiency for error-free TLS in human cells.

PrimPol is a human DNA polymerase-primase that localizes to mitochondria and nucleus and bypasses the major oxidative lesion 7,8-dihydro-8-oxoguanine (oxoG) via translesion synthesis, in mostly error-free manner. We present structures of PrimPol insertion complexes with a DNA template-primer and correct dCTP or erroneous dATP opposite the lesion, as well as extension complexes with C or A as a 3′−terminal primer base. We show that during the insertion of C and extension from it, the active site is unperturbed, reflecting the readiness of PrimPol to accommodate oxoG(anti). The misinsertion of A opposite oxoG(syn) also does not alter the active site, and is likely less favorable due to lower thermodynamic stability of the oxoG(syn)•A base-pair. During the extension step, oxoG(syn) induces an opening of its base-pair with A or misalignment of the 3′-A primer terminus. Together, the structures show how PrimPol accurately synthesizes DNA opposite oxidatively damaged DNA in human cells., The human DNA primase and DNA polymerase PrimPol replicates through the major oxidative DNA damage lesion 7,8-dihydro-8-oxoguanine (oxoG) via translesion synthesis in a mostly error-free manner thus suppressing oxoG-induced mutagenesis in mitochondria and the nucleus. Here, the authors present crystal structures of PrimPol in complex with an oxoG lesion in different contexts that provide mechanistic insights into how PrimPol performs predominantly accurate synthesis on oxidative-damaged DNAs in human cells.

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 a 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.

Chemotherapy with cisplatin becomes limiting due to toxicity and secondary malignancies. In principle, therapeutics could be improved by targeting translesion synthesis (TLS) polymerases (Pols) that promote replication through intrastrand cross-links, the major cisplatin-induced DNA adduct. However, to specifically target malignancies with minimal adverse effects on normal cells, a good understanding of TLS mechanisms in normal versus cancer cells is paramount. We show that in normal cells, TLS through cisplatin intrastrand cross-links is promoted by Polη- or Polι-dependent pathways, both of which require Rev1 as a scaffolding component. In contrast, cancer cells require Rev1-Polζ. Our findings that a recently identified Rev1 inhibitor, JH-RE-06, purported to specifically disrupt Rev1 interaction with Polζ to block TLS through cisplatin adducts in cancer cells, abrogates Rev1's ability to function with Y family Pols as well, implying that by inactivating Rev1-dependent TLS in normal cells, this inhibitor will exacerbate the toxicity and tumorigenicity of chemotherapeutics with cisplatin.

DNA polymerase δ (Polδ) plays pivotal roles in eukaryotic DNA replication and repair. Polδ is conserved from yeast to humans, and mutations in human Polδ have been implicated in various cancers. Saccharomyces cerevisiae Polδ consists of catalytic Pol3 and the regulatory Pol31 and Pol32 subunits. Here, we present the near atomic resolution (3.2 A) cryo-EM structure of yeast Polδ holoenzyme in the act of DNA synthesis. The structure reveals an unexpected arrangement in which the regulatory subunits (Pol31 and Pol32) lie next to the exonuclease domain of Pol3 but do not engage the DNA. The Pol3 C-terminal domain contains a 4Fe−4S cluster and emerges as the keystone of Polδ assembly. We also show that the catalytic and regulatory subunits rotate relative to each other and that this is an intrinsic feature of the Polδ architecture. Collectively, the structure provides a framework for understanding DNA transactions at the replication fork. A unique arrangement of catalytic and regulatory subunits revealed by cryo-EM analysis of DNA polymerase δ holoenzyme in a template–primer complex suggests how interactions between the functional modules promote DNA synthesis and proofreading activities.

As an integral scaffolding component of DNA polymerase (Pol) zeta, Pol lambda adapts Pol zeta–dependent translesion synthesis to operate in a predominantly error-free manner in human cells., 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.

Here we show that translesion synthesis (TLS) opposite 1,N

Cytarabine (AraC) is an essential chemotherapeutic for acute myeloid leukemia (AML) and resistance to this drug is a major cause of treatment failure. AraC is a nucleoside analog that differs from 2′-deoxycytidine only by the presence of an additional hydroxyl group at the C2′ position of the 2′-deoxyribose. The active form of the drug AraC 5′-triphosphate (AraCTP) is utilized by human replicative DNA polymerases to insert AraC at the 3′ terminus of a growing DNA chain. This impedes further primer extension and is a primary basis for the drug action. The Y-family translesion synthesis (TLS) DNA polymerase η (Polη) counteracts this barrier to DNA replication by efficient extension from AraC-terminated primers. Here, we provide high-resolution structures of human Polη with AraC incorporated at the 3′-primer terminus. We show that Polη can accommodate AraC at different stages of the catalytic cycle, and that it can manipulate the conformation of the AraC sugar via specific hydrogen bonding and stacking interactions. Taken together, the structures provide a basis for the ability of Polη to extend DNA synthesis from AraC terminated primers.

Translesion synthesis (TLS) DNA polymerases (Pols) promote replication through DNA lesions; however, little is known about the protein factors that affect their function in human cells. In yeast, Rev1 plays a noncatalytic role as an indispensable component of Polζ, and Polζ together with Rev1 mediates a highly mutagenic mode of TLS. However, how Rev1 functions in TLS and mutagenesis in human cells has remained unclear. Here we determined the role of Rev1 in TLS opposite UV lesions in human and mouse fibroblasts and showed that Rev1 is indispensable for TLS mediated by Polη, Polι, and Polκ but is not required for TLS by Polζ. In contrast to its role in mutagenic TLS in yeast, Rev1 promotes predominantly error-free TLS opposite UV lesions in humans. The identification of Rev1 as an indispensable scaffolding component for Polη, Polι, and Polκ, which function in TLS in highly specialized ways opposite a diverse array of DNA lesions and act in a predominantly error-free manner, implicates a crucial role for Rev1 in the maintenance of genome stability in humans.

Acrolein, an α,β-unsaturated aldehyde, is generated in vivo as the end product of lipid peroxidation and from metabolic oxidation of polyamines, and it is a ubiquitous environmental pollutant. The reaction of acrolein with the N2 of guanine in DNA leads to the formation of γ-hydroxy-1-N(2)-propano-2′ deoxyguanosine (γ-HOPdG), which can exist in DNA in a ring-closed or a ring-opened form. Here, we identified the translesion synthesis (TLS) DNA polymerases (Pols) that conduct replication through the permanently ring-opened reduced form of γ-HOPdG ((r) γ-HOPdG) and show that replication through this adduct is mediated via Rev1/Polη-, Polι/Polκ-, and Polθ-dependent pathways, respectively. Based on biochemical and structural studies, we propose a role for Rev1 and Polι in inserting a nucleotide (nt) opposite the adduct and for Pols η and κ in extending synthesis from the inserted nt in the respective TLS pathway. Based on genetic analyses and biochemical studies with Polθ, we infer a role for Polθ at both the nt insertion and extension steps of TLS. Whereas purified Rev1 and Polθ primarily incorporate a C opposite (r) γ-HOPdG, Polι incorporates a C or a T opposite the adduct; nevertheless, TLS mediated by the Polι-dependent pathway as well as by other pathways occurs in a predominantly error-free manner in human cells. We discuss the implications of these observations for the mechanisms that could affect the efficiency and fidelity of TLS Pols.

N3-Methyladenine (3-MeA) is formed in DNA by reaction with S-adenosylmethionine, the reactive methyl donor, and by reaction with alkylating agents. 3-MeA protrudes into the DNA minor groove and strongly blocks synthesis by replicative DNA polymerases (Pols). However, the mechanisms for replicating through this lesion in human cells remain unidentified. Here we analyzed the roles of translesion synthesis (TLS) Pols in the replication of 3-MeA-damaged DNA in human cells. Because 3-MeA has a short half-life in vitro, we used the stable 3-deaza analog, 3-deaza-3-methyladenine (3-dMeA), which blocks the DNA minor groove similarly to 3-MeA. We found that replication through the 3-dMeA adduct is mediated via three different pathways, dependent upon Polι/Polκ, Polθ, and Polζ. As inferred from biochemical studies, in the Polι/Polκ pathway, Polι inserts a nucleotide (nt) opposite 3-dMeA and Polκ extends synthesis from the inserted nt. In the Polθ pathway, Polθ carries out both the insertion and extension steps of TLS opposite 3-dMeA, and in the Polζ pathway, Polζ extends synthesis following nt insertion by an as yet unidentified Pol. Steady-state kinetic analyses indicated that Polι and Polθ insert the correct nt T opposite 3-dMeA with a much reduced catalytic efficiency and that both Pols exhibit a high propensity for inserting a wrong nt opposite this adduct. However, despite their low fidelity of synthesis opposite 3-dMeA, TLS opposite this lesion replicates DNA in a highly error-free manner in human cells. We discuss the implications of these observations for TLS mechanisms in human cells.

Summary Cancers from sun-exposed skin accumulate “driver” mutations, causally implicated in oncogenesis. Because errors incorporated during translesion synthesis (TLS) opposite UV lesions would generate these mutations, TLS mechanisms are presumed to underlie cancer development. To address the role of TLS in skin cancer formation, we determined which DNA polymerase is responsible for generating UV mutations, analyzed the relative contributions of error-free TLS by Polη and error-prone TLS by Polθ to the replication of UV-damaged DNA and to genome stability, and examined the incidence of UV-induced skin cancers in Polθ−/−, Polη−/−, and Polθ−/− Polη−/− mice. Our findings that the incidence of skin cancers rises in Polθ−/− mice and is further exacerbated in Polθ−/− Polη−/− mice compared with Polη−/− mice support the conclusion that error-prone TLS by Polθ provides a safeguard against tumorigenesis and suggest that cancer formation can ensue in the absence of somatic point mutations.

N1-methyl-deoxyadenosine (1-MeA) is formed by methylation of deoxyadenosine at the N1 atom. 1-MeA presents a block to replicative DNA polymerases due to its inability to participate in Watson-Crick (W-C) base pairing. Here we determine how human DNA polymerase-ι (Polι) promotes error-free replication across 1-MeA. Steady state kinetic analyses indicate that Polι is ~100 fold more efficient in incorporating the correct nucleotide T versus the incorrect nucleotide C opposite 1-MeA. To understand the basis of this selectivity, we determined ternary structures of Polι bound to template 1-MeA and incoming dTTP or dCTP. In both structures, template 1-MeA rotates to the syn conformation but pairs differently with dTTP versus dCTP. Thus, whereas dTTP partakes in stable Hoogsteen base pairing with 1-MeA, dCTP fails to gain a “foothold” and is largely disordered. Together, our kinetic and structural studies show how Polι maintains discrimination between correct and incorrect incoming nucleotide opposite 1-MeA in preserving genome integrity.

Analysis of crystal structure elucidates the mechanism by which a human enzyme acts as both a primase and a DNA polymerase., PrimPol is a novel human enzyme that contains both DNA primase and DNA polymerase activities. We present the first structure of human PrimPol in ternary complex with a DNA template-primer and an incoming deoxynucleoside triphosphate (dNTP). The ability of PrimPol to function as a DNA primase stems from a simple but remarkable feature—almost complete lack of contacts to the DNA primer strand. This, in turn, allows two dNTPs to bind initiation and elongation sites on the enzyme for the formation of the first dinucleotide. PrimPol shows the ability to synthesize DNA opposite ultraviolet (UV) lesions; however, unexpectedly, the active-site cleft of the enzyme is constrained, which precludes the bypass of UV-induced DNA lesions by conventional translesion synthesis. Together, the structure addresses long-standing questions about how DNA primases actually initiate synthesis and how primase and polymerase activities combine in a single enzyme to carry out DNA synthesis.

The Polα/primase complex assembles the short RNA-DNA fragments for priming of lagging and leading strand DNA replication in eukaryotes. As such, the Polα polymerase subunit encounters two types of substrates during primer synthesis: an RNA:DNA helix and a DNA:DNA helix. The engagement of the polymerase subunit with the DNA:DNA helix has been suggested as the of basis for primer termination in eukaryotes. However, there is no structural information on how the Polα polymerase subunit actually engages with a DNA:DNA helix during primer synthesis. We present here the first crystal structure of human Polα polymerase subunit in complex with a DNA:DNA helix. Unexpectedly, we find that portion of the DNA:DNA helix in contact with the polymerase is not in a B-form but in a hybrid A-B form. Almost all of the contacts observed previously with an RNA primer are preserved with a DNA primer – with the same set of polymerase residues tracking the sugar-phosphate backbone of the DNA or RNA primer. Thus, rather than loss of specific contacts, the free energy cost of distorting DNA from B- to hybrid A-B form may augur the termination of primer synthesis in eukaryotes.

A major clinical problem in the use of cisplatin to treat cancers is tumor resistance. DNA polymerase η (Pol-η) is a crucial polymerase that allows cancer cells to cope with the cisplatin-DNA adducts that are formed during chemotherapy. We present here a structure of human Pol-η inserting deoxycytidine triphosphate (dCTP) opposite a cisplatin intrastrand cross-link (PtGpG). We show that the specificity of human Pol-η for PtGpG derives from an active site that is open to permit Watson-Crick geometry of the nascent PtGpG-dCTP base pair and to accommodate the lesion without steric hindrance. This specificity is augmented by the residues Gln38 and Ser62, which interact with PtGpG, and Arg61, which interacts with the incoming dCTP. Collectively, the structure provides a basis for understanding how Pol-η in human cells can tolerate the DNA damage caused by cisplatin chemotherapy and offers a framework for the design of inhibitors in cancer therapy.

In yeast, the Rad6-Rad18 ubiquitin conjugating enzyme plays a critical role in promoting replication although DNA lesions by translesion synthesis (TLS). In striking contrast, a number of studies have indicated that TLS can occur in the absence of Rad18 in human and other mammalian cells, and also in chicken cells. In this study, we determine the role of Rad18 in TLS that occurs during replication in human and mouse cells, and show that in the absence of Rad18, replication of duplex plasmids containing a cis-syn TT dimer or a (6-4) TT photoproduct is severely inhibited in human cells and that mutagenesis resulting from TLS opposite cyclobutane pyrimidine dimers and (6-4) photoproducts formed at the TT, TC, and CC dipyrimidine sites in the chromosomal cII gene in UV-irradiated mouse cells is abolished. From these and other observations with Rad18, we conclude that the Rad6-Rad18 enzyme plays an essential role in promoting replication through DNA lesions by TLS in mammalian cells. In contrast, the dispensability of Rad18 for TLS in chicken DT40 cells would suggest that the role of the Rad6-Rad18 enzyme complex has diverged considerably between chicken and mammals, raising the possibility that TLS mechanisms differ among them.

Exposure of DNA to UV radiation causes covalent linkages between adjacent pyrimidines. The most common lesion found in DNA from these UV-induced linkages is the cis-syn cyclobutane pyrimidine dimer. Human DNA polymerase κ (Polκ), a member of the Y-family of DNA polymerases, is unable to insert nucleotides opposite the 3'T of a cis-syn T-T dimer, but it can efficiently extend from a nucleotide inserted opposite the 3'T of the dimer by another DNA polymerase. We present here the structure of human Polκ in the act of inserting a nucleotide opposite the 5'T of the cis-syn T-T dimer. The structure reveals a constrained active-site cleft that is unable to accommodate the 3'T of a cis-syn T-T dimer but is remarkably well adapted to accommodate the 5'T via Watson-Crick base pairing, in accord with a proposed role for Polκ in the extension reaction opposite from cyclobutane pyrimidine dimers in vivo.

Summary7,8-dihydro-8-oxoguanine (8-oxoG) adducts are formed frequently by the attack of oxygen-free radicals on DNA. They are among the most mutagenic lesions in cells because of their dual coding potential, where, in addition to normal base-pairing of 8-oxoG(anti) with dCTP, 8-oxoG in the syn conformation can base pair with dATP, causing G to T transversions. We provide here for the first time a structural basis for the error-free replication of 8-oxoG lesions by yeast DNA polymerase η (Polη). We show that the open active site cleft of Polη can accommodate an 8-oxoG lesion in the anti conformation with only minimal changes to the polymerase and the bound DNA: at both the insertion and post-insertion steps of lesion bypass. Importantly, the active site geometry remains the same as in the undamaged complex and provides a basis for the ability of Polη to prevent the mutagenic replication of 8-oxoG lesions in cells.

Thymine glycol (Tg) is the most common DNA lesion of thymine induced by interaction with reactive oxygen species. Because of the addition of hydroxyl groups at C5 and C6 in a Tg lesion, the damaged base loses its aromatic character and becomes nonplanar; consequently, the C5 methyl group protrudes in an axial direction and that prevents the stacking of the 5' base above the Tg lesion. Because Tg presents a severe block to continued synthesis by replicative DNA polymerases, we determine here how human cells manage to replicate through this lesion. Using a duplex plasmid system where bidirectional replication ensues from an origin of replication, we show that translesion synthesis (TLS) makes a prominent contribution to Tg bypass and that it occurs in a predominantly error-free fashion. Also, we provide evidence that Pol kappa and Pol zeta function together in promoting error-free replication through the lesion, and based on structural and biochemical information, we propose a role for Pol kappa at the insertion step and of Pol zeta at the extension step of Tg bypass. We discuss the implications of these observations and suggest that human cells have adapted the TLS machinery to function in a much more error-free fashion than could have been predicted from the intrinsic catalytic efficiencies and fidelities of TLS polymerases.

The Rad6-Rad18 mediated monoubiquitylation of proliferating cell nuclear antigen (PCNA) at lys 164 plays a crucial role in promoting the access of translesion synthesis (TLS) DNA polymerases (Pols) to PCNA in the replication fork stalled at a lesion site. Although a number of genetic and biochemical observations have provided strong evidence that TLS Pols bind PCNA at its interdomain connector loop (IDCL) via their PCNA-interacting protein (PIP) domain, a more recent proposal formulates that TLS Pols bind PCNA at two sites, to the IDCL via their PIP domain and to lys-164 linked ubiquitin (Ub) via their ubiquitin-binding domain. To ascertain the relative contributions of the PIP and Ub-binding zinc finger (UBZ) domains of human Polη in TLS, we have determined whether the C-terminal truncations of hPolη that contain the PIP1 domain but lack the UBZ and PIP2 domains can still function in TLS in human cells. Our observations that such C-terminally truncated proteins promote efficient TLS opposite a cis-syn TT dimer and confer a high degree of UV resistance to XPV cells provide unambiguous evidence that the binding of PCNA via its PIP domain is essential as well as sufficient for providing hPolη the ability to carry out TLS in human cells.

The ultraviolet (UV)-induced (6–4) pyrimidine–pyrimidone photoproduct [(6–4) PP] confers a large structural distortion in DNA. Here we examine in human cells the roles of translesion synthesis (TLS) DNA polymerases (Pols) in promoting replication through a (6–4) TT photoproduct carried on a duplex plasmid where bidirectional replication initiates from an origin of replication. We show that TLS contributes to a large fraction of lesion bypass and that it is mostly error-free. We find that, whereas Pol η and Pol ι provide alternate pathways for mutagenic TLS, surprisingly, Pol ζ functions independently of these Pols and in a predominantly error-free manner. We verify and extend these observations in mouse cells and conclude that, in human cells, TLS during replication can be markedly error-free even opposite a highly distorting DNA lesion.

DNA polymerase δ (Polδ) is a high fidelity polymerase that plays a central role in replication from yeast to humans. We present here the crystal structure of the catalytic subunit of yeast Polδ in ternary complex with a template-primer and an incoming nucleotide. The structure, determined at 2.0Å resolution, catches the enzyme in the act of replication. The structure reveals how the polymerase and exonuclease domains are juxtaposed relative to each other and how a correct nucleotide is selected and incorporated. The structure also reveals the “sensing” interactions near the primer terminus that signal a switch from the polymerizing to the editing mode. Taken together, the structure provides a chemical basis for the bulk of DNA synthesis in eukaryotic cells and a framework for understanding the effects of mutations in Polδ̣ that cause cancers.

Treatment of yeast and human cells with DNA-damaging agents elicits Rad6–Rad18-mediated monoubiquitination of proliferating cell nuclear antigen (PCNA) at its Lys-164 residue [ubiquitin (Ub)-PCNA], and this PCNA modification is indispensable for promoting the access of translesion synthesis (TLS) polymerases (Pols) to PCNA. However, the means by which K164-linked Ub modulates the proficiency of TLS Pols to bind PCNA and take over synthesis from the replicative Pol has remained unclear. One model that has gained considerable credence is that the TLS Pols bind PCNA at 2 sites, to the interdomain connector loop via their PCNA-interacting protein (PIP) domain and to the K164-linked Ub moiety via their Ub-binding domain (UBD). Specifically, this model postulates that the UBD-mediated binding of TLS Pols to the Ub moiety on PCNA is necessary for TLS. To test the validity of this model, we examine the contributions that the PIP and Ub-binding zinc finger (UBZ) domains of human Polη make to its functional interaction with PCNA, its colocalization with PCNA in replication foci, and its role in TLS in vivo. We conclude from these studies that the binding to PCNA via its PIP domain is a prerequisite for Polη's ability to function in TLS in human cells and that the direct binding of the Ub moiety on PCNA via its UBZ domain is not required. We discuss the possible role of the Ub moiety on PCNA in TLS.

Acrolein is generated as the end product of lipid peroxidation and is also a ubiquitous environmental pollutant. Its reaction with the N2 of guanine leads to a cyclic gamma-HOPdG adduct that presents a block to normal replication. We show here that yeast Rev1 incorporates the correct nucleotide C opposite a permanently ring-closed form of gamma-HOPdG (PdG) with nearly the same efficiency as opposite an undamaged G. The structural basis of this action lies in the eviction of the PdG adduct from the Rev1 active site, and the pairing of incoming dCTP with a "surrogate" arginine residue. We also show that yeast Polzeta can carry out the subsequent extension reaction. Together, our studies reveal how the exocyclic PdG adduct is accommodated in a DNA polymerase active site, and they show that the combined action of Rev1 and Polzeta provides for accurate and efficient synthesis through this potentially carcinogenic DNA lesion.

In Saccharomyces cerevisiae, replication through DNA lesions is promoted by Rad6-Rad18-dependent processes that include translesion synthesis by DNA polymerases eta and zeta and a Rad5-Mms2-Ubc13-controlled postreplicational repair (PRR) pathway which repairs the discontinuities in the newly synthesized DNA that form opposite from DNA lesions on the template strand. Here, we examine the contributions of the RAD51, RAD52, and RAD54 genes and of the RAD50 and XRS2 genes to the PRR of UV-damaged DNA. We find that deletions of the RAD51, RAD52, and RAD54 genes impair the efficiency of PRR and that almost all of the PRR is inhibited in the absence of both Rad5 and Rad52. We suggest a role for the Rad5 pathway when the lesion is located on the leading strand template and for the Rad52 pathway when the lesion is located on the lagging strand template. We surmise that both of these pathways operate in a nonrecombinational manner, Rad5 by mediating replication fork regression and template switching via its DNA helicase activity and Rad52 via a synthesis-dependent strand annealing mode. In addition, our results suggest a role for the Rad50 and Xrs2 proteins and thereby for the MRX complex in promoting PRR via both the Rad5 and Rad52 pathways.

Nucleotide excision repair (NER) is a versatile DNA repair process which operates on bulky helix-distorting lesions caused by a variety of DNA-damaging agents, including UV and chemical agents. In eukaryotes, following the initial recognition of the DNA lesion by the damage recognition factors, the damaged strand is unwound from the undamaged strand by the DNA helicases present in TFIIH to promote the dual incision of the damaged strand on the 5′ side and the 3′ side of the lesion by two separate endonucleases, which then results in the removal of a fragment of ∼30 nucleotides containing the lesion (21, 25). In eukaryotes, the genetic controls of NER for the nontranscribed regions and the transcribed regions of the genome differ in some important respects. For example, in the yeast Saccharomyces cerevisiae, the RAD7 and RAD16 genes are specifically required for the repair of nontranscribed regions of the genome as well as for the repair of the nontranscribed DNA strand (21). The Rad7 and Rad16 proteins form a heterodimeric complex in which Rad16, a member of the Swi2/Snf2 family of ATPases, provides a DNA-dependent ATPase activity, and the Rad7-Rad16 complex binds preferentially to UV-damaged DNA in an ATP-dependent manner (8, 9). We previously suggested a role for the Rad7-Rad16 complex in the scanning of nontranscribed regions of the genome for DNA lesions and in initiating the nucleation of NER protein factors at the lesion site (8, 9). Rad16 also harbors a C3HC4 motif, a characteristic feature of ubiquitin (Ub) ligases; however, how Rad16 functions in such a role is not understood. DNA lesions from the transcribed strand of expressed genes are removed faster than lesions from the nontranscribed strand, a phenomenon known as transcription-coupled repair (TCR) (17, 18, 29). TCR is conserved from Escherichia coli to humans, and the process in E. coli, in which a transcription repair coupling factor promotes the release of the stalled RNA polymerase along with the nascent transcript from the damage site and then recruits the NER proteins for lesion removal, is well understood (27). In humans, TCR is modulated by Cockayne syndrome A and B proteins (CSA and CSB), and in yeast, TCR is modulated by Rad26, which is the CSB counterpart (21, 25). Although it is generally accepted that the stalled RNA polymerase II (Pol II) has to be removed from the lesion site for the repair machinery to assemble there, it is not known whether Pol II is removed or translocated away from the lesion site or whether it undergoes some sort of conformational change to allow the assembly of NER proteins at the lesion site. Another phenomenon that is observed upon treatment of yeast or human cells with DNA-damaging agents is lysine 48-linked polyubiquitylation of the Rpb1 subunit of Pol II, which targets Pol II for proteasomal degradation (6, 23, 24). It remains unclear, however, whether Pol II polyubiquitylation contributes to TCR by targeting it for degradation, thereby promoting the assembly of the NER proteins at the lesion site. Moreover, since the Ub ligase (E3) responsible for Pol II polyubiquitylation in yeast or human cells has not been identified, this phenomenon has not been amenable to genetic analysis. Our recent observation that elongin C (Elc1) was required for Pol II polyubiquitylation and degradation in yeast cells raised the possibility that an Elc1-dependent Ub ligase might be involved in this process (24). Yeast Elc1 is a homolog of mammalian elongin C (1), which forms a heterotrimeric complex with elongins A and B (3). In mammalian cells, the elongin A, B, and C complex increases the rate of transcription elongation by suppressing Pol II pausing (4, 5). In yeast cells, however, the only elongins present are elongins A and C, and the complex of these two proteins does not stimulate transcription elongation by Pol II (14). Since Elc1 is required for Pol II polyubiquitylation and degradation in yeast cells (24), and since it exists in vivo in a complex with the Rad7 and Rad16 proteins (22), we first considered the possibility that this protein complex was involved in modulating Pol II modification. The Elc1-Rad7-Rad16 complex shares several characteristic features of the SCF (Skp1-Cullin-F box)-type Ub ligases (15). In the SCF complex, the Skp1 subunit acts as an adaptor which connects the F-box protein to the scaffold protein Cdc53/cullin, to which the RING-H2 finger protein Rbx1/Roc1 binds. The F-box protein binds the protein substrates targeted for ubiquitylation by the Ub-conjugating enzyme (E2) which assembles with the Cdc53/cullin subunit. In the Rad7-Rad16-Elc1 complex, Rad7 is an F-box protein, Rad16 is a RING-H2 protein, and Elc1 is a Skp1 homolog. However, our finding that Pol II polyubiquitylation and degradation were not affected in rad7Δ or rad16Δ yeast cells treated with UV or 4-NQO (4-nitroquinoline-1-oxide) (24) led us to consider other possibilities. Here we show that elongin A (Ela1) and cullin 3 (Cul3) are required for Pol II polyubiquitylation and degradation in DNA-damaged yeast cells, and based upon these and other observations, we propose that an E3 comprised of Elc1, Ela1, Cul3, and Roc1 (Rbx1) mediates this process in yeast cells. Further, our genetic analyses indicate that Pol II polyubiquitylation and degradation are not a prerequisite for Rad26-mediated TCR; rather, these two processes provide separate means of lesion removal from the transcribed strand. In addition, these observations lead us to predict that in human cells, the von Hippel-Lindau (VHL) tumor suppressor complex (28, 32), comprised of elongin B, elongin C, cullin 2, and Rbx1 subunits, would function in an analogous manner in mediating Pol II polyubiquitylation and degradation.

Human DNA polymerase kappa (Pol kappa) is a proficient extender of mispaired primer termini on undamaged DNAs and is implicated in the extension step of lesion bypass. We present here the structure of Pol kappa catalytic core in ternary complex with DNA and an incoming nucleotide. The structure reveals encirclement of the DNA by a unique "N-clasp" at the N terminus of Pol kappa, which augments the conventional right-handed grip on the DNA by the palm, fingers, and thumb domains and the PAD and provides additional thermodynamic stability. The structure also reveals an active-site cleft that is constrained by the close apposition of the N-clasp and the fingers domain, and therefore can accommodate only a single Watson-Crick base pair. Together, DNA encirclement and other structural features help explain Pol kappa's ability to extend mismatches and to promote replication through various minor groove DNA lesions, by extending from the nucleotide incorporated opposite the lesion by another polymerase.

N1-methyl adenine (1-MeA) is formed in DNA by reaction with alkylating agents and naturally occurring methyl halides. The 1-MeA lesion impairs Watson-Crick base pairing and blocks normal DNA replication. Here we identify the translesion synthesis (TLS) DNA polymerases (Pols) required for replicating through 1-MeA in human cells and show that TLS through this lesion is mediated via three different pathways in which Pols ι and θ function in one pathway and Pols η and ζ, respectively, function in the other two pathways. Our biochemical studies indicate that in the Polι/Polθ pathway, Polι would carry out nucleotide insertion opposite 1-MeA from which Polθ would extend synthesis. In the Polη pathway, this Pol alone would function at both the nucleotide insertion and extension steps of TLS, and in the third pathway, Polζ would extend from the nucleotide inserted opposite 1-MeA by an as yet unidentified Pol. Whereas by pushing 1-MeA into the syn conformation and by forming Hoogsteen base pair with the T residue, Polι would carry out TLS opposite 1-MeA, the ability of Polη to replicate through 1-MeA suggests that despite its need for Watson-Crick hydrogen bonding, Polη can stabilize the adduct in its active site. Remarkably, even though Pols η and ι are quite error-prone at inserting nucleotides opposite 1-MeA, TLS opposite this lesion in human cells occurs in a highly error-free fashion. This suggests that the in vivo fidelity of TLS Pols is regulated by factors such as post-translational modifications, protein-protein interactions, and possibly others.

Rev1, a Y family DNA polymerase (Pol) functions together with Polzeta, a B family Pol comprised of the Rev3 catalytic subunit and Rev7 accessory subunit, in promoting translesion DNA synthesis (TLS). Extensive genetic studies with Saccharomyces cerevisiae have indicated a requirement of both Polzeta and Rev1 for damage-induced mutagenesis, implicating their involvement in mutagenic TLS. Polzeta is specifically adapted to promote the extension step of lesion bypass, as it proficiently extends primer termini opposite DNA lesions, and it is also a proficient extender of mismatched primer termini on undamaged DNAs. Since TLS through UV-induced lesions and various other DNA lesions does not depend upon the DNA-synthetic activity of Rev1, Rev1 must contribute to Polzeta-dependent TLS in a nonenzymatic way. Here, we provide evidence for the physical association of Rev1 with Polzeta and show that this binding is mediated through the C terminus of Rev1 and the polymerase domain of Rev3. Importantly, a rev1 mutant that lacks the C-terminal 72 residues which inactivate interaction with Rev3 exhibits the same high degree of UV sensitivity and defectiveness in UV-induced mutagenesis as that conferred by the rev1Delta mutation. We propose that Rev1 binding to Polzeta is indispensable for the targeting of Polzeta to the replication fork stalled at a DNA lesion. In addition to this structural role, Rev1 binding enhances the proficiency of Polzeta for the extension of mismatched primer termini on undamaged DNAs and for the extension of primer termini opposite DNA lesions.

Human DNA polymerase kappa (pol kappa) is a member of the Y family of DNA polymerases that function in translesion synthesis. It synthesizes DNA with moderate fidelity and does not efficiently incorporate nucleotides opposite DNA lesions. Pol kappa has the unusual ability to efficiently extend from mismatched primer termini, and it extends readily from nucleotides inserted by other DNA polymerases opposite a variety of DNA lesions. All of this has suggested that pol kappa functions during the extension step of translesion synthesis. Here, we have carried out pre-steady-state kinetic studies of pol kappa using DNA with matched and mismatched primer termini. Interestingly, we find that mismatches present only a modest kinetic barrier to nucleotide incorporation by pol kappa. Moreover, and quite surprisingly, active-site titrations revealed that the concentration of active pol kappa is very low with matched DNA, and from DNA trapping experiments we determined that this was due to the formation of nonproductive protein.DNA complexes. In marked contrast, we found that the concentration of active pol kappa was six-fold greater with mismatched DNA than with matched DNA. Thus, pol kappa forms nonproductive complexes with matched but not with mismatched DNA. From these observations, we conclude that pol kappa has evolved to specifically function on DNA substrates with aberrant primer-terminal base pairs, such as the ones it would encounter during the extension step of translesion synthesis.

Treatment of Saccharomyces cerevisiae and human cells with DNA-damaging agents such as UV light or 4-nitroquinoline-1-oxide induces polyubiquitylation of the largest RNA polymerase II (Pol II) subunit, Rpb1, which results in rapid Pol II degradation by the proteasome. Here we identify a novel role for the yeast Elc1 protein in mediating Pol II polyubiquitylation and degradation in DNA-damaged yeast cells and propose the involvement of a ubiquitin ligase, of which Elc1 is a component, in this process. In addition, we present genetic evidence for a possible involvement of Elc1 in Rad7-Rad16-dependent nucleotide excision repair (NER) of lesions from the nontranscribed regions of the genome and suggest a role for Elc1 in increasing the proficiency of repair of nontranscribed DNA, where as a component of the Rad7-Rad16-Elc1 ubiquitin ligase, it would promote the efficient turnover of the NER ensemble from the lesion site in a Rad23-19S proteasomal complex-dependent reaction.

Nucleotide excision repair (NER) in eukaryotes requires the assembly of a large number of protein factors at the lesion site which then coordinate the dual incision of the damaged DNA strand. However, the manner by which the different protein factors are assembled at the lesion site has remained unclear. Previously, we have shown that in the yeast Saccharomyces cerevisiae, NER proteins exist as components of different protein subassemblies: the Rad1-Rad10 nuclease, for example, forms a tight complex with the damage recognition protein Rad14, and the complex of Rad1-Rad10-Rad14 can be purified intact from yeast cells. As the Rad1-Rad10 nuclease shows no specificity for binding UV lesions in DNA, association with Rad14 could provide an effective means for the targeting of Rad1-Rad10 nuclease to damage sites in vivo. To test the validity of this idea, here we identify two rad1 mutations that render yeast cells as UV sensitive as the rad1Delta mutation but which have no effect on the recombination function of Rad1. From our genetic and biochemical studies with these rad1 mutations, we conclude that the ability of Rad1-Rad10 nuclease to associate in a complex with Rad14 is paramount for the targeting of this nuclease to lesion sites in vivo. We discuss the implications of these observations for the means by which the different NER proteins are assembled at the lesion site.

Human DNA polymerase iota (hPoliota), a member of the Y family of DNA polymerases, differs in remarkable ways from other DNA polymerases, incorporating correct nucleotides opposite template purines with a much higher efficiency and fidelity than opposite template pyrimidines. We present here the crystal structure of hPoliota bound to template G and incoming dCTP, which reveals a G.C + Hoogsteen base pair in a DNA polymerase active site. We show that the hPoliota active site has evolved to favor Hoogsteen base pairing, wherein the template sugar is fixed in a cavity that reduces the C1'-C1' distance across the nascent base pair from approximately 10.5 A in other DNA polymerases to 8.6 A in hPoliota. The rotation of G from anti to syn is then largely in response to this curtailed C1'-C1' distance. A G.C+ Hoogsteen base pair suggests a specific mechanism for hPoliota's ability to bypass N(2)-adducted guanines that obstruct replication.

UV-light-induced cyclobutane pyrimidine dimers (CPDs) present a severe block to synthesis by replicative DNA polymerases (Pols), whereas Polη promotes proficient and error-free replication through CPDs. Although the archael Dpo4, which, like Polη, belongs to the Y family of DNA Pols, can also replicate through a CPD, it is much less efficient than Polη. The x-ray crystal structure of Dpo4 complexed with either the 3′-thymine (T) or the 5′ T of a cis-syn TT dimer has indicated that, whereas the 3′ T of the dimer forms a Watson–Crick base pair with the incoming dideoxy ATP, the 5′ T forms a Hoogsteen base pair with the dideoxy ATP in syn conformation. Based upon these observations, a similar mechanism involving Hoogsteen base pairing of the 5′ T of the dimer with the incoming A has been proposed for Polη. Here we examine the mechanisms of CPD bypass by Dpo4 and Polη using nucleotide analogs that specifically disrupt the Hoogsteen or Watson–Crick base pairing. Our results show that both Dpo4 and Polη incorporate dATP opposite the 5′ T of the CPD via Watson–Crick base pairing and not by Hoogsteen base pairing. Furthermore, opposite the 3′ T of the dimer, the two Pols differ strikingly in the mechanisms of dATP incorporation, with Dpo4 incorporating opposite an abasic-like intermediate and Polη using the normal Watson–Crick base pairing. These observations have important implications for the mechanisms used for the inefficient vs. efficient bypass of CPDs by DNA Pols.

The efficiency and fidelity of nucleotide incorporation by high-fidelity replicative DNA polymerases (Pols) are governed by the geometric constraints imposed upon the nascent base pair by the active site. Consequently, these polymerases can efficiently and accurately replicate through the template bases which are isosteric to natural DNA bases but which lack the ability to engage in Watson-Crick (W-C) hydrogen bonding. DNA synthesis by Poleta, a low-fidelity polymerase able to replicate through DNA lesions, however, is inhibited in the presence of such an analog, suggesting a dependence of this polymerase upon W-C hydrogen bonding. Here we examine whether human Polkappa, which differs from Poleta in having a higher fidelity and which, unlike Poleta, is inhibited at inserting nucleotides opposite DNA lesions, shows less of a dependence upon W-C hydrogen bonding than does Poleta. We find that an isosteric thymidine analog is replicated with low efficiency by Polkappa, whereas a nucleobase analog lacking minor-groove H bonding potential is replicated with high efficiency. These observations suggest that both Poleta and Polkappa rely on W-C hydrogen bonding for localizing the nascent base pair in the active site for the polymerization reaction to occur, thus overcoming these enzymes' low geometric selectivity.