PCNA trimer instability inhibits translesion synthesis by DNA polymerase η and by DNA polymerase δ

Translesion synthesis (TLS), the process by which DNA polymerases replicate through DNA lesions, is the source of most DNA damage-induced mutations. Sometimes TLS is carried out by replicative polymerases that have evolved to synthesize DNA on non-damaged templates. Most of the time, however, TLS is carried out by specialized translesion polymerases that have evolved to synthesize DNA on damaged templates. TLS requires the mono-ubiquitylation of the replication accessory factor proliferating cell nuclear antigen (PCNA). PCNA and ubiquitin-modified PCNA (UbPCNA) stimulate TLS by replicative and translesion polymerases. Two mutant forms of PCNA, one with an E113G substitution and one with a G178S substitution, support normal cell growth but inhibit TLS thereby reducing mutagenesis in yeast. A re-examination of the structures of both mutant PCNA proteins revealed substantial disruptions of the subunit interface that forms the PCNA trimer. Both mutant proteins have reduced trimer stability with the G178S substitution causing a more severe defect. The mutant forms of PCNA and UbPCNA do not stimulate TLS of an abasic site by either replicative Pol δ or translesion Pol η. Normal replication by Pol η was also impacted, but normal replication by Pol δ was much less affected. These findings support a model in which reduced trimer stability causes these mutant PCNA proteins to occasionally undergo conformational changes that compromise their ability to stimulate TLS by both replicative and translesion polymerases.     

DNA polymerases eta and kappa exchange with the polymerase delta holoenzyme to complete common fragile site synthesis

Common fragile sites (CFSs) are inherently unstable genomic loci that are recurrently altered in human tumor cells. Despite their instability, CFS are ubiquitous throughout the human genome and associated with large tumor suppressor genes or oncogenes. CFSs are enriched with repetitive DNA sequences, one feature postulated to explain why these loci are inherently difficult to replicate, and sensitive to replication stress. We have shown that specialized DNA polymerases (Pols) η and κ replicate CFS-derived sequences more efficiently than the replicative Pol δ. However, we lacked an understanding of how these enzymes cooperate to ensure efficient CFS replication. Here, we designed a model of lagging strand replication with RFC loaded PCNA that allows for maximal activity of the four-subunit human Pol δ holoenzyme, Pol η, and Pol κ in polymerase mixing assays. We discovered that Pol η and κ are both able to exchange with Pol δ stalled at repetitive CFS sequences, enhancing Normalized Replication Efficiency. We used this model to test the impact of PCNA mono-ubiquitination on polymerase exchange, and found no change in polymerase cooperativity in CFS replication compared with unmodified PCNA. Finally, we modeled replication stress in vitro using aphidicolin and found that Pol δ holoenzyme synthesis was significantly inhibited in a dose-dependent manner, preventing any replication past the CFS. Importantly, Pol η and κ were still proficient in rescuing this stalled Pol δ synthesis, which may explain, in part, the CFS instability phenotype of aphidicolin-treated Pol η and Pol κ-deficient cells. In total, our data support a model wherein Pol δ stalling at CFSs allows for free exchange with a specialized polymerase that is not driven by PCNA.     

Translesion synthesis across abasic lesions by human B-family and Y-family DNA polymerases α, δ, η, ι, κ, and REV1

Abasic (apurinic/apyrimidinic, AP) sites are the most common DNA lesions formed in cells, induce severe blocks to DNA replication, and are highly mutagenic. Human Y-family translesion DNA polymerases (pols) such as pols η, ι, κ, and REV1 have been suggested to play roles in replicative bypass across many DNA lesions where B-family replicative pols stall, but their individual catalytic functions in AP site bypass are not well understood. In this study, oligonucleotides containing a synthetic abasic lesion (tetrahydrofuran analogue) were compared for catalytic efficiency and base selectivity with human Y-family pols η, ι, κ, and REV1 and B-family pols α and δ. Pol η and pol δ/proliferating cell nuclear antigen (PCNA) copied past AP sites quite effectively and generated products ranging from one-base to full-length extension. Pol ι and REV1 readily incorporated one base opposite AP sites but then stopped. Pols κ and α were severely blocked at AP sites. Pol η preferentially inserted T and A; pol ι inserted T, G, and A; pol κ inserted C and A; REV1 preferentially inserted C opposite AP sites. The B-family pols α and δ/PCNA preferentially inserted A (85% and 58%, respectively) consonant with the A-rule hypothesis. Pols η and δ/PCNA were much more efficient in next-base extension, preferably from A positioned opposite an AP site, than pol κ. These results suggest that AP sites might be bypassed with moderate efficiency by single B- and Y-family pols or combinations, possibly by REV1 and pols ι, η, and δ/PCNA at the insertion step opposite the lesion and by pols η and δ/PCNA at the subsequent extension step. The patterns of the base preferences of human B-family and Y-family pols in both insertion and extension are pertinent to some of the mutagenesis events induced by AP lesions in human cells.     

A novel variant of DNA polymerase ζ, Rev3ΔC,highlights differential regulation of Pol32 as a subunit of polymerase δ versus ζ in Saccharomyces cerevisiae

Unrepaired DNA lesions often stall replicative DNA polymerases and are bypassed by translesion synthesis (TLS) to prevent replication fork collapse. Mechanisms of TLS are lesion- and species-specific, with a prominent role of specialized DNA polymerases with relaxed active sites. After nucleotide(s) are incorporated across from the altered base(s), the aberrant primer termini are typically extended by DNA polymerase ζ (pol ζ). As a result, pol ζ is responsible for most DNA damage-induced mutations. The mechanisms of sequential DNA polymerase switches in vivo remain unclear. The major replicative DNA polymerase δ (pol δ) shares two accessory subunits, called Pol31/Pol32 in yeast, with pol ζ. Inclusion of Pol31/Pol32 in the pol δ/pol ζ holoenzymes requires a [4Fe–4S] cluster in C-termini of the catalytic subunits. Disruption of this cluster in Pol ζ or deletion of POL32 attenuates induced mutagenesis. Here we describe a novel mutation affecting the catalytic subunit of pol ζ, rev3ΔC, which provides insight into the regulation of pol switches. Strains with Rev3ΔC, lacking the entire C-terminal domain and therefore the platform for Pol31/Pol32 binding, are partially proficient in Pol32-dependent UV-induced mutagenesis. This suggests an additional role of Pol32 in TLS, beyond being a pol ζ subunit, related to pol δ. In search for members of this regulatory pathway, we examined the effects of Maintenance of Genome Stability 1 (Mgs1) protein on mutagenesis in the absence of Rev3–Pol31/Pol32 interaction. Mgs1 may compete with Pol32 for binding to PCNA. Mgs1 overproduction suppresses induced mutagenesis, but had no effect on UV-mutagenesis in the rev3ΔC strain, suggesting that Mgs1 exerts its inhibitory effect by acting specifically on Pol32 bound to pol ζ. The evidence for differential regulation of Pol32 in pol δ and pol ζ emphasizes the complexity of polymerase switches.     

The ADP-ribosyltransferase PARP10/ARTD10 Interacts with Proliferating Cell Nuclear Antigen (PCNA) and Is Required for DNA Damage Tolerance

All cells rely on genomic stability mechanisms to protect against DNA alterations. PCNA is a master regulator of DNA replication and S-phase-coupled repair. PCNA post-translational modifications by ubiquitination and SUMOylation dictate how cells stabilize and re-start replication forks stalled at sites of damaged DNA. PCNA mono-ubiquitination recruits low fidelity DNA polymerases to promote error-prone replication across DNA lesions. Here, we identify the mono-ADP-ribosyltransferase PARP10/ARTD10 as a novel PCNA binding partner. PARP10 knockdown results in genomic instability and DNA damage hypersensitivity. Importantly, we show that PARP10 binding to PCNA is required for translesion DNA synthesis. Our work identifies a novel PCNA-linked mechanism for genome protection, centered on post-translational modification by mono-ADP-ribosylation.     

Proliferating cell unclear antigen-associated factor (PAF15): A novel oncogene

Proliferating cell nuclear antigen (PCNA)-Associated Factor (PAF15) is a small protein containing a PCNA interacting motif and sequences for association with ubiquitin enzymes. In interaction with PCNA, PAF15 plays a key role in recruiting DNA replicative polymerase by double monoubiquitination at Lys¹⁵ and Lys²⁴. Under DNA damage conditions, PAF15 regulates the switch from DNA replicative polymerase to translesion synthesis polymerase in order to bypass the replication-blocking lesions. Overexpression of PAF15 promotes the repair of ultraviolet-induced DNA damage and prevents cell death, whereas attenuation of PAF15 decreases DNA replication and cell survival. Ectopic expression of PAF15 in mouse fibroblasts increases colony formation and tumourigenicity. PAF15 is aberrantly increased in various human malignancies with poor prognosis. Collectively, PAF15 may contribute to carcinogenesis and represents one of the potential therapeutic targets in the treatment of cancer.     

Translesion synthesis: Y-family polymerases and the polymerase switch

Replicative DNA polymerases are blocked at DNA lesions. Synthesis past DNA damage requires the replacement of the replicative polymerase by one of a group of specialised translesion synthesis (TLS) polymerases, most of which belong to the Y-family. Each of these has different substrate specificities for different types of damage. In eukaryotes mono-ubiquitination of PCNA plays a crucial role in the switch from replicative to TLS polymerases at stalled forks. All the Y-family polymerases have ubiquitin binding sites that increase their binding affinity for ubiquitinated PCNA at the sites of stalled forks.     

Proliferating Cell Nuclear Antigen (PCNA)-binding Protein C1orf124 Is a Regulator of Translesion Synthesis

DNA damage-induced proliferating cell nuclear antigen (PCNA) ubiquitination serves as the key event mediating post-replication repair. Post-replication repair involves either translesion synthesis (TLS) or damage avoidance via template switching. In this study, we have identified and characterized C1orf124 as a regulator of TLS. C1orf124 co-localizes and interacts with unmodified and mono-ubiquitinated PCNA at UV light-induced damage sites, which require the PIP box and UBZ domain of C1orf124. C1orf124 also binds to the AAA-ATPase valosin-containing protein via its SHP domain, and cellular resistance to UV radiation mediated by C1orf124 requires its interactions with valosin-containing protein and PCNA. Interestingly, C1orf124 binds to replicative DNA polymerase POLD3 and PDIP1 under normal conditions but preferentially associates with TLS polymerase η (POLH) upon UV damage. Depletion of C1orf124 compromises PCNA monoubiquitination, RAD18 chromatin association, and RAD18 localization to UV damage sites. Thus, C1orf124 acts at multiple steps in TLS, stabilizes RAD18 and ubiquitinated PCNA at damage sites, and facilitates the switch from replicative to TLS polymerase to bypass DNA lesion.     

In vitro gap-directed translesion DNA synthesis of an abasic site involving human DNA polymerases epsilon, lambda, and beta

DNA polymerase (pol) ε is thought to be the leading strand replicase in eukaryotes, whereas pols λ and β are believed to be mainly involved in re-synthesis steps of DNA repair. DNA elongation by the human pol ε is halted by an abasic site (apurinic/apyrimidinic (AP) site). In this study, we present in vitro evidence that human pols λ, β, and η can perform translesion synthesis (TLS) of an AP site in the presence of pol ε, likely by initiating the 3'OHs created at the lesion by the arrested pol ε. However, in the case of pols λ and β, this TLS requires the presence of a DNA gap downstream from the product synthesized by the pol ε, and the optimal gap for efficient TLS is different for the two polymerases. The presence of gaps did not affect the TLS capacity of human pol η. Characterization of the reaction products showed that pol β inserted dAMP opposite the AP site, whereas gap filling synthesis by pol λ resulted in single or double deletions opposite the lesion. The synthesis up to the AP site by pol ε and the subsequent TLS by pols λ and β are not influenced by human processivity factor proliferating cell nuclear antigen and human single-stranded DNA-binding protein replication protein A. The bypass capacity of pol λ at the AP site is greatly reduced when a truncated form of the enzyme, which has lost the BRCA1 C-terminal and proline-rich domains, is used. Collectively, our in vitro results support the existence of a mechanism of gap-directed TLS at an AP site involving a switch between the replicative pol ε and the repair pols λ and β.     

DNA polymerase δ and ζ switch by sharing accessory subunits of DNA polymerase δ

Translesion DNA synthesis is an important branch of the DNA damage tolerance pathway that assures genomic integrity of living organisms. The mechanisms of DNA polymerase (Pol) switches during lesion bypass are not known. Here, we show that the C-terminal domain of the Pol ζ catalytic subunit interacts with accessory subunits of replicative DNA Pol δ. We also show that, unlike other members of the human B-family of DNA polymerases, the highly conserved and similar C-terminal domains of Pol δ and Pol ζ contain a [4Fe-4S] cluster coordinated by four cysteines. Amino acid changes in Pol ζ that prevent the assembly of the [4Fe-4S] cluster abrogate Pol ζ function in UV mutagenesis. On the basis of these data, we propose that Pol switches at replication-blocking lesions occur by the exchange of the Pol δ and Pol ζ catalytic subunits on a preassembled complex of accessory proteins retained on DNA during translesion DNA synthesis.     

Beyond translesion synthesis: polymerase κ fidelity as a potential determinant of microsatellite stability

Microsatellite DNA synthesis represents a significant component of human genome replication that must occur faithfully. However, yeast replicative DNA polymerases do not possess high fidelity for microsatellite synthesis. We hypothesized that the structural features of Y-family polymerases that facilitate accurate translesion synthesis may promote accurate microsatellite synthesis. We compared human polymerases κ (Pol κ) and η (Pol η) fidelities to that of replicative human polymerase δ holoenzyme (Pol δ4), using the in vitro HSV-tk assay. Relative polymerase accuracy for insertion/deletion (indel) errors within 2-3 unit repeats internal to the HSV-tk gene concurred with the literature: Pol δ4 > Pol κ or Pol η. In contrast, relative polymerase accuracy for unit-based indel errors within [GT](10) and [TC](11) microsatellites was: Pol κ ≥ Pol δ4 > Pol η. The magnitude of difference was greatest between Pols κ and δ4 with the [GT] template. Biochemically, Pol κ displayed less synthesis termination within the [GT] allele than did Pol δ4. In dual polymerase reactions, Pol κ competed with either a stalled or moving Pol δ4, thereby reducing termination. Our results challenge the ideology that pol κ is error prone, and suggest that DNA polymerases with complementary biochemical properties can function cooperatively at repetitive sequences.     

Roles of POLD4, smallest subunit of DNA polymerase δ, in nuclear structures and genomic stability of human cells

Mammalian DNA polymerase δ (pol δ) is essential for DNA replication, though the functions of this smallest subunit of POLD4 have been elusive. We investigated pol δ activities in vitro and found that it was less active in the absence of POLD4, irrespective of the presence of the accessory protein PCNA. shRNA-mediated reduction of POLD4 resulted in a marked decrease in colony formation activity by Calu6, ACC-LC-319, and PC-10 cells. We also found that POLD4 reduction was associated with an increased population of karyomere-like cells, which may be an indication of DNA replication stress and/or DNA damage. The karyomere-like cells retained an ability to progress through the cell cycle, suggesting that POLD4 reduction induces modest genomic instability, while allowing cells to grow until DNA damage reaches an intolerant level. Our results indicate that POLD4 is required for the in vitro pol δ activity, and that it functions in cell proliferation and maintenance of genomic stability of human cells.     

Crystal Structure of SUMO-Modified Proliferating Cell Nuclear Antigen

Eukaryotic proliferating cell nuclear antigen (PCNA) is a replication accessory protein that functions in DNA replication, repair, and recombination. The various functions of PCNA are regulated by posttranslational modifications including mono-ubiquitylation, which promotes translesion synthesis, and sumoylation, which inhibits recombination. To understand how SUMO modification regulates PCNA, we generated a split SUMO-modified PCNA protein and showed that it supports cell viability and stimulates DNA polymerase δ activity. We then determined its X-ray crystal structure and found that SUMO occupies a position on the back face of the PCNA ring, which is distinct from the position occupied by ubiquitin in the structure of ubiquitin-modified PCNA. We propose that the back of PCNA has evolved to be a site of regulation that can be easily modified without disrupting ongoing reactions on the front of PCNA, such as normal DNA replication. Moreover, these modifications likely allow PCNA to function as a tool belt, whereby proteins can be recruited to the replication machinery via the back of PCNA and be held in reserve until needed.     

Translesion synthesis in mammalian cells

DNA damage blocks the progression of the replication fork. In order to circumvent the damaged bases, cells employ specialized low stringency DNA polymerases, which are able to carry out translesion synthesis (TLS) past different types of damage. The five polymerases used in TLS in human cells have different substrate specificities, enabling them to deal with many different types of damaged bases. PCNA plays a central role in recruiting the TLS polymerases and effecting the polymerase switch from replicative to TLS polymerase. When the fork is blocked PCNA gets ubiquitinated. This increases its affinity for the TLS polymerases, which all have novel ubiquitin-binding motifs, thereby facilitating their engagement at the stalled fork to effect TLS.     

Structure of monoubiquitinated PCNA

Ubiquitination of proliferating cell nuclear antigen (PCNA) to ub-PCNA is essential for DNA replication across bulky template lesions caused by UV radiation and alkylating agents, as ub-PCNA orchestrates the recruitment and switching of translesion synthesis (TLS) polymerases with replication polymerases. This allows replication to proceed, leaving the DNA to be repaired subsequently. Defects in a TLS polymerase, Pol η, lead to a form of Xeroderma pigmentosum, a disease characterized by severe skin sensitivity to sunlight damage and an increased incidence of skin cancer. Structurally, however, information on how ub-PCNA orchestrates the switching of these two classes of polymerases is lacking. We have solved the structure of ub-PCNA and demonstrate that the ubiquitin molecules in ub-PCNA are radially extended away from the PCNA without structural contact aside from the isopeptide bond linkage. This unique orientation provides an open platform for the recruitment of TLS polymerases through ubiquitin-interacting domains. However, the ubiquitin moieties, to the side of the equatorial PCNA plane, can place spatial constraints on the conformational flexibility of proteins bound to ub-PCNA. We show that ub-PCNA is impaired in its ability to support the coordinated actions of Fen1 and Pol δ in assays mimicking Okazaki fragment processing. This provides evidence for the novel concept that ub-PCNA may modulate additional DNA transactions other than TLS polymerase recruitment and switching.     

Oxidative DNA damage bypass in Arabidopsis thaliana requires DNA polymerase λ and proliferating cell nuclear antigen 2

The oxidized base 7,8-oxoguanine (8-oxo-G) is the most common DNA lesion generated by reactive oxygen species. This lesion is highly mutagenic due to the frequent misincorporation of A opposite 8-oxo-G during DNA replication. In mammalian cells, the DNA polymerase (pol) family X enzyme DNA pol λ catalyzes the correct incorporation of C opposite 8-oxo-G, together with the auxiliary factor proliferating cell nuclear antigen (PCNA). Here, we show that Arabidopsis thaliana DNA pol λ, the only member of the X family in plants, is as efficient in performing error-free translesion synthesis past 8-oxo-G as its mammalian homolog. Arabidopsis, in contrast with animal cells, possesses two genes for PCNA. Using in vitro and in vivo approaches, we observed that PCNA2, but not PCNA1, physically interacts with DNA pol λ, enhancing its fidelity and efficiency in translesion synthesis. The levels of DNA pol λ in transgenic plantlets characterized by overexpression or silencing of Arabidopsis POLL correlate with the ability of cell extracts to perform error-free translesion synthesis. The important role of DNA pol λ is corroborated by the observation that the promoter of POLL is activated by UV and that both overexpressing and silenced plants show altered growth phenotypes.     

Error-free bypass of 2-hydroxyadenine by human DNA polymerase lambda with Proliferating Cell Nuclear Antigen and Replication Protein A in different sequence contexts

1,2-dihydro-2-oxoadenine (2-OH-A), a common DNA lesion produced by reactive oxygen species, is a strong replicative block for several DNA polymerases (DNA pols). We have previously shown that various bases can be misincorporated opposite the 2-OH-A lesion and the type of mispairs varies with either the sequence context or the type of DNA pol tested. Here, we have analysed the ability of the human pol family X member DNA pol λ, to bypass the 2-OH-A lesion. DNA pol λ can perform error-free bypass of 2-OH-A when this lesion is located in a random sequence, whereas in a repeated sequence context, even though bypass was also largely error-free, misincorporation of dGMP could be observed. The fidelity of translesion synthesis of 2-OH-A in a repeated sequence by DNA pol λ was enhanced by the auxiliary proteins Proliferating Cell Nuclear Antigen (PCNA) and Replication Protein A (RP-A). We also found that the DNA pol λ active site residue tyrosine 505 determined the nucleotide selectivity opposite 2-OH-A. Our data show, for the first time, that the 2-OH-A lesion can be efficiently and faithfully bypassed by a human DNA pol λ in combination with PCNA and RP-A.     

DNA polymerase δ and ɛ holoenzymes from calf thymus

Replication of singly-DNA primed M13 DNA by DNA polymerase (pol) δ completely relies on the simultaneous addition of proliferating cell nuclear antigen (PCNA), replication factor C (RF-C) and replication protein A (RP-A) (orE.coli singlestrand DNA binding protein, SSB). Pol ? core alone is able to synthesize the products on singly-primed ssDNA. However, DNA synthesis by pol ? was stimulated up to 10-fold upon addition of the three auxiliary proteins PCNA, RF-C and SSB. This stimulation of pol ? by PCNA/RF-C/SSB appears to be the superposition of two events: pol, ? holoenzyme (pol ?, PCNA, RF-C) synthesized longer products than its pol ? core counterpart, but elongated less primers. Furthermore, we analyzed the cooperative action of pol α/primase with pol δ or pol ? holoenzymes on unprimed M13 DNA. While pol δ displayed higher dNMP incorporation than pol ?, when a single primer was preannealed to DNA, pol ? was more efficient in the utilization of the primers synthesized by pol α/primase. Under these conditions both longer products and a higher amount of dNMP incorporation was found for pol ? holoenzyme, than for pol δ. Our data support the hypothesis of pol δ as the leading and pol ? as the second lagging strand replication enzyme.     

Rad18-mediated Translesion Synthesis of Bulky DNA Adducts Is Coupled to Activation of the Fanconi Anemia DNA Repair Pathway

Fanconi anemia (FA) is a cancer susceptibility syndrome characterized by sensitivity to DNA-damaging agents. The FA proteins (FANCs) are implicated in DNA repair, although the precise mechanisms by which FANCs process DNA lesions are not fully understood. An epistatic relationship between the FA pathway and translesion synthesis (TLS, a post-replication DNA repair mechanism) has been suggested, but the basis for cross-talk between the FA and TLS pathways is poorly understood. We show here that ectopic overexpression of the E3 ubiquitin ligase Rad18 (a central regulator of TLS) induces DNA damage-independent mono-ubiquitination of proliferating cell nuclear antigen (PCNA) (a known Rad18 substrate) and FANCD2. Conversely, DNA damage-induced mono-ubiquitination of both PCNA and FANCD2 is attenuated in Rad18-deficient cells, demonstrating that Rad18 contributes to activation of the FA pathway. WT Rad18 but not an E3 ubiquitin ligase-deficient Rad18 C28F mutant fully complements both PCNA ubiquitination and FANCD2 activation in Rad18-depleted cells. Rad18-induced mono-ubiquitination of FANCD2 is not observed in FA core complex-deficient cells, demonstrating that Rad18 E3 ligase activity alone is insufficient for FANCD2 ubiquitylation. Instead, Rad18 promotes FA core complex-dependent FANCD2 ubiquitination in a manner that is secondary to PCNA mono-ubiquitination. Taken together, these results demonstrate a novel Rad18-dependent mechanism that couples activation of the FA pathway with TLS.     

Ubiquitination of PCNA and the Polymerase Switch in Human Cells

Replicative DNA polymerases are blocked by damage in the template DNA. To get past this damage, the cell employs specialised translesion synthesis (TLS) polymerases, which have reduced stringency and are able to bypass different lesions. For example, DNA polymerase ? (pol?) is able to carry out TLS past UV-induced cyclobutane pyrimidine dimers. How does the cell bring about the switch from replicative to TLS polymerase? We have shown that, in human cells, when the replication machinery is blocked at DNA damage, PCNA, the sliding clamp required for DNA replication, is mono-ubiquitinated and that this modified form of PCNA has increased affinity for pol?. This provides a mechanism for the polymerase switch. In this Extra-View, we discuss the possible signals that might trigger ubiquitination of PCNA, whether PCNA becomes de-ubiquitinated after TLS has been accomplished and the role of the hREV1 protein in TLS. We point out some apparent differences between mechanisms in Saccharomyces cerevisiae and human cells.