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.     

PTIP/Swift is required for efficient PCNA ubiquitination in response to DNA damage

Monoubiquitination of proliferating cell nuclear antigen (PCNA) enables translesion synthesis (TLS) by specialized DNA polymerases to replicate past damaged DNA. We have studied PCNA modification and chromatin recruitment of TLS polymerases in Xenopus egg extracts and mammalian cells. We show that Xenopus PCNA becomes ubiquitinated and sumoylated after replication stress induced by UV or aphidicolin. Under these conditions the TLS polymerase eta was recruited to chromatin and also became monoubiquitinated. PTIP/Swift is an adaptor protein for the ATM/ATR kinases. Immunodepletion of PTIP/Swift from Xenopus extracts prevented efficient PCNA ubiquitination and polymerase eta recruitment to chromatin during replicative stress. In addition to PCNA ubiquitination, efficient polymerase eta recruitment to chromatin also required ATR kinase activity. We also show that PTIP depletion from mammalian cells by RNAi reduced PCNA ubiquitination in response to DNA damage, and also decreased the recruitment to chromatin of polymerase eta and the recombination protein Rad51. Our results suggest that PTIP/Swift is an important new regulator of DNA damage avoidance in metazoans.     

Structural insight into recruitment of translesion DNA polymerase Dpo4 to sliding clamp PCNA

DNA polymerases are co-ordinated by sliding clamps (PCNA/β-clamp) in translesion synthesis. It is unclear how these enzymes assemble on PCNA with geometric and functional compatibility. We report the crystal structure of a full-length Y-family polymerase, Dpo4, in complex with heterodimeric PCNA1–PCNA2 at 2.05 Å resolution. Dpo4 exhibits an extended conformation that differs from the Dpo4 structures in apo- or DNA-bound form. Two hinges have been identified in Dpo4, which render the multidomain polymerase flexible conformations and orientations relative to PCNA. Dpo4 binds specifically to PCNA1 on the conserved ligand binding site. The C-terminal peptide of Dpo4 becomes structured with a 3₁₀ helix and dominates the specific binding. The Y-family polymerase also contacts PCNA1 with its finger, thumb and little finger domains, which are conformation-dependent protein–protein interactions that diversify the binding mode of Dpo4 on PCNA. The structure reveals a molecular model in which substrate/partner binding-coupled multiple conformations of a Y-family polymerase facilitate its recruitment and co-ordination on the sliding clamp. The conformational flexibility would turn the error-prone Y-family polymerase off when more efficient high-fidelity DNA polymerases work on undamaged DNA and turn it onto DNA templates to perform translesion synthesis when replication forks are stalled by DNA lesions.     

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.     

Intrinsic Flexibility of Ubiquitin on Proliferating Cell Nuclear Antigen (PCNA) in Translesion Synthesis

Ubiquitin conjugation provides a crucial signaling role in hundreds of cellular pathways; however, a structural understanding of ubiquitinated substrates is lacking. One important substrate is monoubiquitinated PCNA (PCNA-Ub), which signals for recruitment of damage-tolerant polymerases in the translesion synthesis (TLS) pathway of DNA damage avoidance. We use a novel and efficient enzymatic method to produce PCNA-Ub at high yield with a native isopeptide bond and study its Usp1/UAF1-dependent deconjugation. In solution we find that the ubiquitin moiety is flexible relative to the PCNA, with its hydrophobic patch mostly accessible for recruitment of TLS polymerases, which promotes the interaction with polymerase η. The studies are a prototype for the nature of the ubiquitin modification.     

Regulation of translesion DNA synthesis: Posttranslational modification of lysine residues in key proteins

Posttranslational modification of proteins often controls various aspects of their cellular function. Indeed, over the past decade or so, it has been discovered that posttranslational modification of lysine residues plays a major role in regulating translesion DNA synthesis (TLS) and perhaps the most appreciated lysine modification is that of ubiquitination. Much of the recent interest in ubiquitination stems from the fact that proliferating cell nuclear antigen (PCNA) was previously shown to be specifically ubiquitinated at K164 and that such ubiquitination plays a key role in regulating TLS. In addition, TLS polymerases themselves are now known to be ubiquitinated. In the case of human polymerase η, ubiquitination at four lysine residues in its C-terminus appears to regulate its ability to interact with PCNA and modulate TLS. Within the past few years, advances in global proteomic research have revealed that many proteins involved in TLS are, in fact, subject to a previously underappreciated number of lysine modifications. In this review, we will summarize the known lysine modifications of several key proteins involved in TLS; PCNA and Y-family polymerases η, ι, κ and Rev1 and we will discuss the potential regulatory effects of such modification in controlling TLS in vivo.     

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.     

Proliferating cell nuclear antigen-dependent coordination of the biological functions of human DNA polymerase iota

Y-family DNA polymerases are believed to facilitate the replicative bypass of damaged DNA in a process commonly referred to as translesion synthesis. With the exception of DNA polymerase eta (poleta), which is defective in humans with the Xeroderma pigmentosum variant (XP-V) phenotype, little is known about the cellular function(s) of the remaining human Y-family DNA polymerases. We report here that an interaction between human DNA polymerase iota (poliota) and the proliferating cell nuclear antigen (PCNA) stimulates the processivity of poliota in a template-dependent manner in vitro. Mutations in one of the putative PCNA-binding motifs (PIP box) of poliota or the interdomain connector loop of PCNA diminish the binding between poliota and PCNA and concomitantly reduce PCNA-dependent stimulation of poliota activity. Furthermore, although retaining its capacity to interact with poleta in vivo, the poliota-PIP box mutant fails to accumulate in replication foci. Thus, PCNA, acting as both a scaffold and a modulator of the different activities involved in replication, appears to recruit and coordinate replicative and translesion DNA synthesis polymerases to ensure genome integrity.     

A ubiquitin-binding motif in the translesion DNA polymerase Rev1 mediates its essential functional interaction with ubiquitinated proliferating cell nuclear antigen in response to DNA damage

During normal DNA replication, the proliferating cell nuclear antigen (PCNA) enhances the processivity of DNA polymerases at the replication fork. When DNA damage is encountered, PCNA is monoubiquitinated on Lys-164 by the Rad6-Rad18 complex as the initiating step of translesion synthesis. DNA damage bypass by the translesion synthesis polymerase Rev1 is enhanced by the presence of ubiquitinated PCNA. Here we have carried out a mutational analysis of Rev1, and we have identified the functional domain in the C terminus of Rev1 that mediates interactions with PCNA. We show that a unique motif within this domain binds the ubiquitin moiety of ubiquitinated PCNA. Point mutations within this ubiquitin-binding motif of Rev1 (L821A,P822A,I825A) abolish its functional interaction with ubiquitinated PCNA in vitro and strongly attenuate damage-induced mutagenesis in vivo. Taken together, these studies suggest a specific mechanism by which the interaction between Rev1 and ubiquitinated PCNA is stabilized during the DNA damage response.     

Structural basis for recruitment of translesion DNA polymerase Pol IV/DinB to the beta-clamp

Y-family DNA polymerases can extend primer strands across template strand lesions that stall replicative polymerases. The poor processivity and fidelity of these enzymes, key to their biological role, requires that their access to the primer-template junction is both facilitated and regulated in order to minimize mutations. These features are believed to be provided by interaction with processivity factors, beta-clamp or proliferating cell nuclear antigen (PCNA), which are also essential for the function of replicative DNA polymerases. The basis for this interaction is revealed by the crystal structure of the complex between the 'little finger' domain of the Y-family DNA polymerase Pol IV and the beta-clamp processivity factor, both from Escherichia coli. The main interaction involves a C-terminal peptide of Pol IV, and is similar to interactions seen between isolated peptides and other processivity factors. However, this first structure of an entire domain of a binding partner with an assembled clamp reveals a substantial secondary interface, which maintains the polymerase in an inactive orientation, and may regulate the switch between replicative and Y-family DNA polymerases in response to a template strand lesion.     

Structural basis of ubiquitin recognition by translesion synthesis DNA polymerase ι

Cells have evolved mutagenic bypass mechanisms that prevent stalling of the replication machinery at DNA lesions. This process, translesion DNA synthesis (TLS), involves switching from high-fidelity DNA polymerases to specialized DNA polymerases that replicate through a variety of DNA lesions. In eukaryotes, polymerase switching during TLS is regulated by the DNA damage-triggered monoubiquitylation of PCNA. How the switch operates is unknown, but all TLS polymerases of the so-called Y-family possess PCNA and ubiquitin-binding domains that are important for their function. To gain insight into the structural mechanisms underlying the regulation of TLS by ubiquitylation, we have probed the interaction of ubiquitin with a conserved ubiquitin-binding motif (UBM2) of Y-family polymerase Polι. Using NMR spectroscopy, we have determined the structure of a complex of human Polι UBM2 and ubiquitin, revealing a novel ubiquitin recognition fold consisting of two α-helices separated by a central trans-proline residue conserved in all UBMs. We show that, different from the majority of ubiquitin complexes characterized to date, ubiquitin residue Ile44 only plays a modest role in the association of ubiquitin with Polι UBM2. Instead, binding of UBM2 is centered on the recognition of Leu8 in ubiquitin, which is essential for the interaction.     

Ubiquitination of PCNA and Its Essential Role in Eukaryotic Translesion Synthesis

Ubiquitin and ubiquitin-like proteins (Ubls) are now at the center stage of molecular and cell biology because of their diverse functions in many fundamentally important cellular processes. Besides the celebrated role of ubiquitin in the 26S proteasome-mediated protein degradation pathway, the non-proteolytic functions of ubiquitin are being uncovered at a fast pace. The prominent examples include membrane trafficking, innate immunity, kinase signaling, chromatin dynamics and DNA damage response. Researchers in the area of DNA damage response have witnessed rapid progress within the past decade, largely stimulated by the seminal findings that ubiquitination and SUMOylation of a key DNA replication/repair protein, proliferating cell nuclear antigen (PCNA), controls precisely how eukaryotic cells respond to different types of DNA damage, and how cellular DNA damage repair or tolerance pathways are selected to cope with damage in the DNA genome. Here, we will review the recent findings on translesion synthesis (TLS) and its regulation by PCNA ubiquitination in eukaryotes. We will discuss two prevalent models, i.e., the postreplicative gap-filling and the polymerase switch, which have been invoked to account for eukaryotic cells' ability to overcome DNA damage associated replication blockade through TLS. Results from both in vitro reconstitution and from genetic systems will be discussed. We will also summarize the recent findings revealing the crosstalk between two major human DNA damage response pathways (the TLS and the Fanconi anemia pathways), and the ATR and ATM-independent regulation of PCNA ubiquitination. Lastly, new methods of preparing ubiquitinated PCNA will be reviewed. The availability of milligram levels of ubiquitinated PCNA will help our understanding of the molecular details in eukaryotic TLS.     

Effect of proliferating cell nuclear antigen ubiquitination and chromatin structure on the dynamic properties of the Y-family DNA polymerases

Y-family DNA polymerases carry out translesion synthesis past damaged DNA. DNA polymerases (pol) η and ι are usually uniformly distributed through the nucleus but accumulate in replication foci during S phase. DNA-damaging treatments result in an increase in S phase cells containing polymerase foci. Using photobleaching techniques, we show that polη is highly mobile in human fibroblasts. Even when localized in replication foci, it is only transiently immobilized. Although ubiquitination of proliferating cell nuclear antigen (PCNA) is not required for the localization of polη in foci, it results in an increased residence time in foci. polι is even more mobile than polη, both when uniformly distributed and when localized in foci. Kinetic modeling suggests that both polη and polι diffuse through the cell but that they are transiently immobilized for ~150 ms, with a larger proportion of polη than polι immobilized at any time. Treatment of cells with DRAQ5, which results in temporary opening of the chromatin structure, causes a dramatic immobilization of polη but not polι. Our data are consistent with a model in which the polymerases are transiently probing the DNA/chromatin. When DNA is exposed at replication forks, the polymerase residence times increase, and this is further facilitated by the ubiquitination of PCNA.     

Diverse roles of RAD18 and Y-family DNA polymerases in tumorigenesis

Mutagenesis is a hallmark and enabling characteristic of cancer cells. The E3 ubiquitin ligase RAD18 and its downstream effectors, the ‘Y-family’ Trans-Lesion Synthesis (TLS) DNA polymerases, confer DNA damage tolerance at the expense of DNA replication fidelity. Thus, RAD18 and TLS polymerases are attractive candidate mediators of mutagenesis and carcinogenesis. The skin cancer-propensity disorder xeroderma pigmentosum-variant (XPV) is caused by defects in the Y-family DNA polymerase Pol eta (Polη). However it is unknown whether TLS dysfunction contributes more generally to other human cancers. Recent analyses of cancer genomes suggest that TLS polymerases generate many of the mutational signatures present in diverse cancers. Moreover biochemical studies suggest that the TLS pathway is often reprogrammed in cancer cells and that TLS facilitates tolerance of oncogene-induced DNA damage. Here we review recent evidence supporting widespread participation of RAD18 and the Y-family DNA polymerases in the different phases of multi-step carcinogenesis.     

Mismatch repair protein MSH2 regulates translesion DNA synthesis following exposure of cells to UV radiation

Translesion DNA synthesis (TLS) can use specialized DNA polymerases to insert and/or extend nucleotides across lesions, thereby limiting stalled replication fork collapse and the potential for cell death. Recent studies have shown that monoubiquitinated proliferating cell nuclear antigen (PCNA) plays an important role in recruitment of Y-family TLS polymerases to stalled replication forks after DNA damage treatment. To explore the possible roles of other factors that regulate the ultraviolet (UV)-induced assembly of specialized DNA polymerases at arrested replication forks, we performed immunoprecipitation experiments combined with mass spectrometry and established that DNA polymerase kappa (Polκ) can partner with MSH2, an important mismatch repair protein associated with hereditary non-polyposis colorectal cancer. We found that depletion of MSH2 impairs PCNA monoubiquitination and the formation of foci containing Polκ and other TLS polymerases after UV irradiation of cells. Interestingly, expression of MSH2 in Rad18-deficient cells increased UV-induced Polκ and REV1 focus formation without detectable changes in PCNA monoubiquitination, indicating that MSH2 can regulate post-UV focus formation by specialized DNA polymerases in both PCNA monoubiquitination-dependent and -independent fashions. Moreover, we observed that MSH2 can facilitate TLS across cyclobutane pyrimidine dimers photoproducts in living cells, presenting a novel role of MSH2 in post-UV cellular responses.     

Epstein-Barr virus BPLF1 deubiquitinates PCNA and attenuates polymerase η recruitment to DNA damage sites

PCNA is monoubiquitinated in response to DNA damage and fork stalling and then initiates recruitment of specialized polymerases in the DNA damage tolerance pathway, translesion synthesis (TLS). Since PCNA is reported to associate with Epstein-Barr virus (EBV) DNA during its replication, we investigated whether the EBV deubiquitinating (DUB) enzyme encoded by BPLF1 targets ubiquitinated PCNA and disrupts TLS. An N-terminal BPLF1 fragment (a BPLF1 construct containing the first 246 amino acids [BPLF1 1-246]) associated with PCNA and attenuated its ubiquitination in response to fork-stalling agents UV and hydroxyurea in cultured cells. Moreover, monoubiquitinated PCNA was deubiquitinated after incubation with purified BPLF1 1-246 in vitro. BPLF1 1-246 dysregulated TLS by reducing recruitment of the specialized repair polymerase polymerase η (Polη) to the detergent-resistant chromatin compartment and virtually abolished localization of Polη to nuclear repair foci, both hallmarks of TLS. Expression of BPLF1 1-246 decreased viability of UV-treated cells and led to cell death, presumably through deubiquitination of PCNA and the inability to repair damaged DNA. Importantly, deubiquitination of PCNA could be detected endogenously in EBV-infected cells in comparison with samples expressing short hairpin RNA (shRNA) against BPLF1. Further, the specificity of the interaction between BPLF1 and PCNA was dependent upon a PCNA-interacting peptide (PIP) domain within the N-terminal region of BPLF1. Both DUB activity and PIP sequence are conserved in the members of the family Herpesviridae. Thus, deubiquitination of PCNA, normally deubiquitinated by cellular USP1, by the viral DUB can disrupt repair of DNA damage by compromising recruitment of TLS polymerase to stalled replication forks. PCNA is the first cellular target identified for BPLF1 and its deubiquitinating activity.     

PCNA modifications for regulation of post-replication repair pathways

Stalled DNA replication forks activate specific DNA repair mechanism called post-replication repair (PRR) pathways that simply bypass DNA damage. The bypassing of DNA damage by PRR prevents prolonged stalling of DNA replication that could result in double strand breaks (DSBs). Proliferating cell nuclear antigen (PCNA) functions to initiate and choose different bypassing pathways of PRR. In yeast, DNA replication forks stalled by DNA damage induces monoubiquitination of PCNA at K164, which is catalyzed by Rad6/Rad18 complex. PCNA monoubiquitination triggers the replacement of replicative polymerase with special translesion synthesis (TLS) polymerases that are able to replicate past DNA lesions. The PCNA interaction motif and/or the ubiquitin binding motif in most TLS polymerases seem to be important for the regulation of TLS. The TLS pathway is usually error-prone because TLS polymerases have low fidelity and no proofreading activity. PCNA can also be further polyubiquitinated by Ubc13/ Mms2/Rad5 complex, which adds an ubiquitin chain onto monoubiquitinated K164 of PCNA. PCNA polyubiquitination directs a different PRR pathway known as error-free damage avoidance, which uses the newly synthesized sister chromatid as a template to bypass DNA damage presumably through template switching mechanism. Mammalian homologues of all of the yeast PRR proteins have been identified, thus PRR is well conserved throughout evolution. Mutations of some PRR genes are associated with a higher risk for cancers in mice and human patients, strongly supporting the importance of PRR as a tumor suppressor pathway.     

DNA聚合酶Polι的研究进展

Y家族DNA聚合酶是一种跨损伤复制酶,即能以损伤的DNA为模板进行复制。Y家族DNA聚合酶广泛分布生物界,人类细胞中Y家族DNA聚合酶至少包括Rev1、Polκ、Polι、Polη四种,Polι在以DNA为模板进行复制时错配率很高而不同于其他跨损伤DNA聚合酶,Polι是目前发现的所有DNA聚合酶中保真性最低的DNA聚合酶。很高的错配率导致很高的突变率,最后基因的突变导致癌症的发生,因此Polι在各个国家被广泛的研究,并且对Polι的各个不同的特性进行了研究,取得了一系列成果,现对Polι的研究进展予以综述,并展望了未来的研究趋势。     

ATR-mediated phosphorylation of DNA polymerase η is needed for efficient recovery from UV damage

DNA polymerase η (polη) belongs to the Y-family of DNA polymerases and facilitates translesion synthesis past UV damage. We show that, after UV irradiation, polη becomes phosphorylated at Ser601 by the ataxia-telangiectasia mutated and Rad3-related (ATR) kinase. DNA damage-induced phosphorylation of polη depends on its physical interaction with Rad18 but is independent of PCNA monoubiquitination. It requires the ubiquitin-binding domain of polη but not its PCNA-interacting motif. ATR-dependent phosphorylation of polη is necessary to restore normal survival and postreplication repair after ultraviolet irradiation in xeroderma pigmentosum variant fibroblasts, and is involved in the checkpoint response to UV damage. Taken together, our results provide evidence for a link between DNA damage-induced checkpoint activation and translesion synthesis in mammalian cells.     

Variations on a theme: Eukaryotic Y-family DNA polymerases

Most classical DNA polymerases, which function in normal DNA replication and repair, are unable to synthesize DNA opposite damage in the template strand. Thus in order to replicate through sites of DNA damage, cells are equipped with a variety of nonclassical DNA polymerases. These nonclassical polymerases differ from their classical counterparts in at least two important respects. First, nonclassical polymerases are able to efficiently incorporate nucleotides opposite DNA lesions while classical polymerases are generally not. Second, nonclassical polymerases synthesize DNA with a substantially lower fidelity than do classical polymerases. Many nonclassical polymerases are members of the Y-family of DNA polymerases, and this article focuses on the mechanisms of the four eukaryotic members of this family: polymerase eta, polymerase kappa, polymerase iota, and the Rev1 protein. We discuss the mechanisms of these enzymes at the kinetic and structural levels with a particular emphasis on how they accommodate damaged DNA substrates. Work over the last decade has shown that the mechanisms of these nonclassical polymerases are fascinating variations of the mechanism of the classical polymerases. The mechanisms of polymerases eta and kappa represent rather minor variations, while the mechanisms of polymerase iota and the Rev1 protein represent rather major variations. These minor and major variations all accomplish the same goal: they allow the nonclassical polymerases to circumvent the problems posed by the template DNA lesion.