Localization of DNA polymerases eta and iota to the replication machinery is tightly co-ordinated in human cells

Y-family DNA polymerases can replicate past a variety of damaged bases in vitro but, with the exception of DNA polymerase eta (poleta), which is defective in xeroderma pigmentosum variants, there is little information on the functions of these polymerases in vivo. Here, we show that DNA polymerase iota (poliota), like poleta, associates with the replication machinery and accumulates at stalled replication forks following DNA-damaging treatment. We show that poleta and poliota foci form with identical kinetics and spatial distributions, suggesting that localization of these two polymerases is tightly co-ordinated within the nucleus. Furthermore, localization of poliota in replication foci is largely dependent on the presence of poleta. Using several different approaches, we demonstrate that poleta and poliota interact with each other physically and that the C-terminal 224 amino acids of poliota are sufficient for both the interaction with poleta and accumulation in replication foci. Our results provide strong evidence that poleta targets poliota to the replication machinery, where it may play a general role in maintaining genome integrity as well as participating in translesion DNA synthesis.     

Localization of DNA polymerases eta and iota to the replication machinery is tightly co-ordinated in human cells

Y-family DNA polymerases can replicate past a variety of damaged bases in vitro but, with the exception of DNA polymerase eta (poleta), which is defective in xeroderma pigmentosum variants, there is little information on the functions of these polymerases in vivo. Here, we show that DNA polymerase iota (poliota), like poleta, associates with the replication machinery and accumulates at stalled replication forks following DNA-damaging treatment. We show that poleta and poliota foci form with identical kinetics and spatial distributions, suggesting that localization of these two polymerases is tightly co-ordinated within the nucleus. Furthermore, localization of poliota in replication foci is largely dependent on the presence of poleta. Using several different approaches, we demonstrate that poleta and poliota interact with each other physically and that the C-terminal 224 amino acids of poliota are sufficient for both the interaction with poleta and accumulation in replication foci. Our results provide strong evidence that poleta targets poliota to the replication machinery, where it may play a general role in maintaining genome integrity as well as participating in translesion DNA synthesis.     

Interaction of human DNA polymerase eta with monoubiquitinated PCNA: a possible mechanism for the polymerase switch in response to DNA damage

Most types of DNA damage block replication fork progression during DNA synthesis because replicative DNA polymerases are unable to accommodate altered DNA bases in their active sites. To overcome this block, eukaryotic cells employ specialized translesion synthesis (TLS) polymerases, which can insert nucleotides opposite damaged bases. In particular, TLS by DNA polymerase eta (poleta) is the major pathway for bypassing UV photoproducts. How the cell switches from replicative to TLS polymerase at the site of blocked forks is unknown. We show that, in human cells, PCNA becomes monoubiquitinated following UV irradiation of the cells and that this is dependent on the hRad18 protein. Monoubiquitinated PCNA but not unmodified PCNA specifically interacts with poleta, and we have identified two motifs in poleta that are involved in this interaction. Our findings provide an attractive mechanism by which monoubiquitination of PCNA might mediate the polymerase switch.     

Human DNA polymerase eta promotes DNA synthesis from strand invasion intermediates of homologous recombination

Stalled replication forks pose a serious threat to genome integrity. To overcome the catastrophic consequences associated with fork demise, translesion synthesis (TLS) polymerases such as poleta promote DNA synthesis past lesions. Alternatively, a stalled fork may collapse and undergo repair by homologous recombination. By using fractionated cell extracts and purified recombinant proteins, we show that poleta extends DNA synthesis from D loop recombination intermediates in which an invading strand serves as the primer. Extracts from XP-V cells, which are defective in poleta, exhibit severely reduced D loop extension activity. The D loop extension activity of poleta is unusual, as this reaction cannot be promoted by the replicative DNA polymerase delta or by other TLS polymerases such as poliota. Moreover, we find that poleta interacts with RAD51 recombinase and RAD51 stimulates poleta-mediated D loop extension. Our results indicate a dual function for poleta at stalled replication forks: the promotion of translesion synthesis and the reinitiation of DNA synthesis by homologous recombination repair.     

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.     

Controlling the subcellular localization of DNA polymerases iota and eta via interactions with ubiquitin

Y-family DNA polymerases have spacious active sites that can accommodate a wide variety of geometric distortions. As a consequence, they are considerably more error-prone than high-fidelity replicases. It is hardly surprising, therefore, that the in vivo activity of these polymerases is tightly regulated, so as to minimize their inadvertent access to primer-termini. We report here that one such mechanism employed by human cells relies on a specific and direct interaction between DNA polymerases iota and eta with ubiquitin (Ub). Indeed, we show that both polymerases interact noncovalently with free polyUb chains, as well as mono-ubiquitinated proliferating cell nuclear antigen (Ub-PCNA). Mutants of poliota (P692R) and poleta (H654A) were isolated that are defective in their interactions with polyUb and Ub-PCNA, whilst retaining their ability to interact with unmodified PCNA. Interestingly, the polymerase mutants exhibit significantly lower levels of replication foci in response to DNA damage, thereby highlighting the biological importance of the polymerase-Ub interaction in regulating the access of the TLS polymerases to stalled replication forks in vivo.     

Xeroderma pigmentosum variant and error-prone DNA polymerases

Replicative DNA synthesis is a faithful event which requires undamaged DNA and high fidelity DNA polymerases. If unrepaired damage remains in the template DNA during replication, specialised low fidelity DNA polymerases synthesises DNA past lesions (translesion synthesis, TLS). Current evidence suggests that the polymerase switch from replicative to translesion polymerases might be mediated by post-translational modifications involving ubiquitination processes. One of these TLS polymerases, polymerase eta carries out TLS past UV photoproducts and is deficient in the variant form of xeroderma pigmentosum (XP-V). The dramatic proneness to skin cancer of XP-V individuals highlights the importance of this DNA polymerase in cancer avoidance. The UV hypermutability of XP-V cells suggests that, in the absence of a functional poleta, UV-induced lesions are bypassed by inaccurate DNA polymerase(s) which remain to be identified.     

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 DNA synthesis across non-DNA segments in cultured human cells

DNA lesions that have escaped DNA repair are tolerated via translesion DNA synthesis (TLS), carried out by specialized error-prone DNA polymerases. To evaluate the robustness of the TLS system in human cells, we examined its ability to cope with foreign non-DNA stretches of 3 or 12 methylene residues, using a gap-lesion plasmid assay system. We found that both the trimethylene and dodecamethylene inserts were bypassed with significant efficiencies in human cells, using both misinsertion and misalignment mechanisms. TLS across these non-DNA segments was aphidicolin-sensitive, and did not require poleta. In vitro primer extension assays showed that purified poleta, polkappa and poliota were each capable of inserting each of the four nucleotides opposite the trimethylene chain, but only poleta and polkappa could fully bypass it. Poleta and poliota, but not polkappa, could also insert each of the four nucleotides opposite the dodecamethylene chain, but all three polymerases were severely blocked by this lesion. The ability of TLS polymerases to insert nucleotides opposite a hydrocarbon chain, despite the lack of any similarity to DNA, suggests that they may act via a mode of transient and local template-independent polymerase activity, and highlights the robustness of the TLS system in human cells.     

Ubiquitin-family modifications in the replication of DNA damage

The cell uses specialised Y-family DNA polymerases or damage avoidance mechanisms to replicate past damaged sites in DNA. These processes are under complex regulatory systems, which employ different types of post-translational modification. All the Y-family polymerases have ubiquitin binding domains that bind to mono-ubiquitinated PCNA to effect the switching from replicative to Y-family polymerase. Ubiquitination and de-ubiquitination of PCNA are tightly regulated. There is also evidence for another as yet unidentified ubiquitinated protein being involved in recruitment of Y-family polymerases to chromatin. Poly-ubiquitination of PCNA stimulates damage avoidance, and, at least in yeast, PCNA is SUMOylated to prevent unwanted recombination events at the replication fork. The Y-family polymerases themselves can be ubiquitinated and, in the case of DNA polymerase η, this results in the polymerase being excluded from chromatin.     

DNA Replication-Coupled PCNA Mono-Ubiquitination and Polymerase Switching in a Human InVitro System

Translesion DNA synthesis is a mechanism of DNA damage tolerance, and mono-ubiquitination of proliferating cell nuclear antigen (PCNA) is considered to play a key role in regulating the switch from replicative to translesion DNA polymerases (pols). In this study, we analyzed effects of a replicative pol δ on PCNA mono-ubiquitination with the ubiquitin-conjugating enzyme and ligase UBE2A/HHR6A/RAD6A-RAD18. The results revealed that PCNA interacting with pol δ is a better target for ubiquitination, and PCNA mono-ubiquitination could be coupled with DNA replication. Consequently, we could reconstitute replication-coupled switching between pol δ and a translesion pol, pol η, on an ultraviolet-light-irradiated template. With this system, we obtained direct evidence that polymerase switching reactions are stimulated by mono-ubiquitination of PCNA, depending on a function of the ubiquitin binding zinc finger domain of pol η. This study provides a framework for detailed analyses of molecular mechanisms of human pol switching and regulation of translesion DNA synthesis.     

Replication of damaged DNA by translesion synthesis in human cells

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

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.     

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.     

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.     

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.     

Rad18 guides poleta to replication stalling sites through physical interaction and PCNA monoubiquitination

The DNA replication machinery stalls at damaged sites on templates, but normally restarts by switching to a specialized DNA polymerase(s) that carries out translesion DNA synthesis (TLS). In human cells, DNA polymerase eta (poleta) accumulates at stalling sites as nuclear foci, and is involved in ultraviolet (UV)-induced TLS. Here we show that poleta does not form nuclear foci in RAD18(-/-) cells after UV irradiation. Both Rad18 and Rad6 are required for poleta focus formation. In wild-type cells, UV irradiation induces relocalization of Rad18 in the nucleus, thereby stimulating colocalization with proliferating cell nuclear antigen (PCNA), and Rad18/Rad6-dependent PCNA monoubiquitination. Purified Rad18 and Rad6B monoubiquitinate PCNA in vitro. Rad18 associates with poleta constitutively through domains on their C-terminal regions, and this complex accumulates at the foci after UV irradiation. Furthermore, poleta interacts preferentially with monoubiquitinated PCNA, but poldelta does not. These results suggest that Rad18 is crucial for recruitment of poleta to the damaged site through protein-protein interaction and PCNA monoubiquitination.     

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.     

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.     

Proliferating cell nuclear antigen promotes translesion synthesis by DNA polymerase zeta

DNA polymerase zeta (Pol zeta), a heterodimer of Rev3 and Rev7, is essential for DNA damage provoked mutagenesis in eukaryotes. DNA polymerases that function in a processive complex with the replication clamp proliferating cell nuclear antigen (PCNA) have been shown to possess a close match to the consensus PCNA-binding motif QxxLxxFF. This consensus motif is lacking in either subunit of Pol zeta, yet its activity is stimulated by PCNA. In particular, translesion synthesis of UV damage-containing DNA is dramatically stimulated by PCNA such that translesion synthesis rates are comparable with replication rates by Pol zeta on undamaged DNA. PCNA also stimulated translesion synthesis of a model abasic site by Pol zeta. Efficient PCNA stimulation required that PCNA was prevented from sliding off the damage-containing model oligonucleotide template-primer through the use of biotin-streptavidin bumpers or other blocks. Under those experimental conditions, facile bypass of the abasic site was also detected by DNA polymerase delta or eta (Rad30). The yeast DNA damage checkpoint clamp, consisting of Rad17, Mec3, and Ddc1, and an ortholog of human 9-1-1, has been implicated in damage-induced mutagenesis. However, this checkpoint clamp did not stimulate translesion synthesis by Pol zeta or by DNA polymerase delta.