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.     

Srs2 mediates PCNA‐SUMO‐dependent inhibition of DNA repair synthesis

Completion of DNA replication needs to be ensured even when challenged with fork progression problems or DNA damage. PCNA and its modifications constitute a molecular switch to control distinct repair pathways. In yeast, SUMOylated PCNA (S‐PCNA) recruits Srs2 to sites of replication where Srs2 can disrupt Rad51 filaments and prevent homologous recombination (HR). We report here an unexpected additional mechanism by which S‐PCNA and Srs2 block the synthesis‐dependent extension of a recombination intermediate, thus limiting its potentially hazardous resolution in association with a cross‐over. This new Srs2 activity requires the SUMO interaction motif at its C‐terminus, but neither its translocase activity nor its interaction with Rad51. Srs2 binding to S‐PCNA dissociates Polδ and Polη from the repair synthesis machinery, thus revealing a novel regulatory mechanism controlling spontaneous genome rearrangements. Our results suggest that cycling cells use the Siz1‐dependent SUMOylation of PCNA to limit the extension of repair synthesis during template switch or HR and attenuate reciprocal DNA strand exchanges to maintain genome stability.     

Replication protein A-directed unloading of PCNA by the Ctf18 cohesion establishment complex

The replication clamp PCNA is loaded around DNA by replication factor C (RFC) and functions in DNA replication and repair. Regulated unloading of PCNA during the progression and termination of DNA replication may require additional factors. Here we show that a Saccharomyces cerevisiae complex required for the establishment of sister chromatid cohesion functions as an efficient unloader of PCNA. Unloading requires ATP hydrolysis. This seven-subunit Ctf18-RFC complex consists of the four small subunits of RFC, together with Ctf18, Dcc1, and Ctf8. Ctf18-RFC was also a weak loader of PCNA onto naked template-primer DNA. However, when the single-stranded DNA template was coated by the yeast single-stranded DNA binding protein replication protein A (RPA) but not by a mutant form of RPA or a heterologous single-stranded DNA binding protein, both binding of Ctf18-RFC to substrate DNA and loading of PCNA were strongly inhibited, and unloading predominated. Neither yeast RFC itself nor two other related clamp loaders, containing either Rad24 or Elg1, catalyzed significant unloading of PCNA. The Dcc1 and Ctf8 subunits of Ctf18-RFC, while required for establishing sister chromatid cohesion in vivo, did not function specifically in PCNA unloading in vitro, thereby separating the functionality of the Ctf18-RFC complex into two distinct paths.     

Dynamics of some postreplication DNA repair proteins in carcinogen-damaged mammalian cells

Many types of DNA lesions in template strands block DNA replication and lead to a stalling of replication forks. This block can be overcome (bypassed) by special DNA polymerases (for example, DNA polymerase eta, Pol eta) that perform translesion synthesis on damaged template DNA. The phenomenon of completing DNA replication, while DNA lesions remain in the template strands, has been named post-replication repair (PRR). In yeast Saccharomyces cerevisiae, PRR includes mutagenic and error-free pathways under the regulation of the RAD6/RAD18 complex, which induces ubiquitylation of PCNA. In mammalian cells, Pol eta accumulates in replication foci but the mechanism of this accumulation is not known. Pol eta possesses a conserved PCNA binding motif at the C terminal and phosphorylation of this motif might be essential for its interaction with PCNA. We have shown previously that staurosporine, an inhibitor of protein kinases, inhibits PRR in human cells. In this study we examined whether the accumulation of Pol eta in replication foci after DNA damage is dependent on phosphorylation of the PCNA binding motif. We also studied DNA damage-induced phosphorylation of GFP-tagged human Rad18 (hRad18) and its accumulation in replication foci. Our data indicate that (1) Pol eta is not phosphorylated in response to UV irradiation or MMS treatment, but its diffusional mobility is slightly decreased, and (2) hRad18 accumulates in MMS-treated cells, and considerable amount of the protein co-localizes with detergent insoluble PCNA in replication foci; these responses are sensitive to staurosporine. Our data suggest that hRad18 phosphorylation is the staurosporine-sensitive PRR step.     

Human SHPRH suppresses genomic instability through proliferating cell nuclear antigen polyubiquitination

Differential modifications of proliferating cell nuclear antigen (PCNA) determine DNA repair pathways at stalled replication forks. In yeast, PCNA monoubiquitination by the ubiquitin ligase (E3) yRad18 promotes translesion synthesis (TLS), whereas the lysine-63-linked polyubiquitination of PCNA by yRad5 (E3) promotes the error-free mode of bypass. The yRad5-dependent pathway is important to prevent genomic instability during replication, although its exact molecular mechanism is poorly understood. This mechanism has remained totally elusive in mammals because of the lack of apparent RAD5 homologues. We report that a putative tumor suppressor gene, SHPRH, is a human orthologue of yeast RAD5. SHPRH associates with PCNA, RAD18, and the ubiquitin-conjugating enzyme UBC13 (E2) and promotes methyl methanesulfonate (MMS)-induced PCNA polyubiquitination. The reduction of SHPRH by stable short hairpin RNA increases sensitivity to MMS and enhances genomic instability. Therefore, the yRad5/SHPRH-dependent pathway is a conserved and fundamental DNA repair mechanism that protects the genome from genotoxic stress.     

Damage-specific modification of PCNA

Okazaki fragment processing is an integral part of DNA replication. For a long time, we assumed that the maturation of these small RNA-primed DNA fragments did not necessarily have to occur during S phase, but could be postponed to late in S phase after the bulk of DNA synthesis had been completed. This view was primarily based on the arrest phenotype of temperature-sensitive DNA ligase I mutants in yeast, which accumulated with an almost fully duplicated set of chromosomes. However, many temperature-sensitive alleles can be leaky and the re-evaluation of DNA ligase I-deficient cells has offered new and unexpected insights into how cells keep track of lagging strand synthesis. It turns out that if Okazaki fragment joining goes awry, cells have their own alarm system in the form of ubiquitin that is conjugated to the replication clamp PCNA. Although this modification results in mono- and poly-ubiquitination of PCNA, it is genetically distinct from the known post-replicative repair mark at lysine 164. In this Extra View, we discuss the possibility that eukaryotic cells utilize different enzymatic pathways and ubiquitin attachment sites on PCNA to alert the replication machinery to the accumulation of single-stranded gaps or nicks behind the fork.Key words: DNA ligase I, DNA replication, Okazaki fragment processing, PCNA, ubiquitin, SUMO     

Functional analysis of subcellular localization and protein-protein interaction sequences in the essential DNA ligase I protein of fission yeast

DNA ligase I (Lig I) has key roles in chromosomal DNA replication and repair in the eukaryotic cell nucleus. In the budding yeast Saccharomyces cerevisiae the Lig I enzyme Cdc9p is also required for mitochondrial DNA replication and repair. In this report, dual nuclear–mitochondrial localization is demonstrated to be a property of the essential Lig I enzyme Cdc17 from the distantly related fission yeast Schizosaccharomyces pombe. Expression of nuclear and mitochondrial forms of Cdc17 from separate genes shows that, whereas expression of either protein alone is insufficient to restore viability to cells lacking endogenous Cdc17, co-expression restores full viability. In the nucleus, Lig I interacts with the sliding clamp proliferating cell nuclear antigen (PCNA) via a conserved PCNA interacting sequence motif known as a PIP box. Deletion of the PIP motif from the N-terminus of the nuclear form of Cdc17 fails to abolish Cdc17 function, indicating that PCNA binding by Cdc17 is not an absolute requirement for completion of S-phase.     

Damage-specific modification of PCNA

Okazaki fragment processing is an integral part of DNA replication. For a long time, we assumed that the maturation of these small RNA-primed DNA fragments did not necessarily have to occur during S phase, but could be postponed to late in S phase after the bulk of DNA synthesis had been completed. This view was primarily based on the arrest phenotype of temperature-sensitive DNA ligase I mutants in yeast, which accumulated with an almost fully duplicated set of chromosomes. However, many temperature-sensitive alleles can be leaky, and the re-evaluation of DNA ligase I-deficient cells has offered new and unexpected insights into how cells keep track of lagging strand synthesis. It turns out that if Okazaki fragment joining goes awry, cells have their own alarm system in the form of ubiquitin that is conjugated to the replication clamp PCNA. Although this modification results in mono- and poly-ubiquitination of PCNA, it is genetically distinct from the known post-replicative repair mark at lysine 164. In this Extra View, we discuss the possibility that eukaryotic cells utilize different enzymatic pathways and ubiquitin attachment sites on PCNA to alert the replication machinery to the accumulation of single-stranded gaps or nicks behind the fork.     

Arabidopsis thaliana: Proliferating cell nuclear antigen 1 and 2 possibly form homo- and hetero-trimeric complexes in the plant cell

The proliferating cell nuclear antigen (PCNA) is a key component of the eukaryotic DNA replication machinery. It also plays an important role in DNA repair mechanisms. Despite the intense scientific research on yeast and human PCNA, information describing the function of this protein in plants is still very limited. In the previous study Arabidopsis PCNA2 but not PCNA1 was proposed to be functionally important in DNA polymerase η-dependent postreplication repair. In addition to the above study, PCNA2 but not PCNA1 was also shown to be necessary for Arabidopsis DNA polymerase λ-dependent oxidative DNA damage bypass. Taking into account the reported differences between PCNA1 and PCNA2, we tested the idea of a possible cooperation between PCNA1 and PCNA2 in the plant cell. In a bimolecular fluorescence complementation assay an interaction between PCNA1 and PCNA2 was observed in the nucleus, as well as in the cytoplasm. This finding, together with our previous results, indicates that PCNA1 and PCNA2 may cooperate in planta by forming homo- and heterotrimeric rings. The observed interaction might be relevant when distinct functions for PCNA1 and PCNA2 are considered.     

Recruitment of DNA repair synthesis machinery to sites of DNA damage/repair in living human cells

The eukaryotic sliding DNA clamp, proliferating cell nuclear antigen (PCNA), is essential for DNA replication and repair synthesis. In order to load the ring-shaped, homotrimeric PCNA onto the DNA double helix, the ATPase activity of the replication factor C (RFC) clamp loader complex is required. Although the recruitment of PCNA by RFC to DNA replication sites has well been documented, our understanding of its recruitment during DNA repair synthesis is limited. In this study, we analyzed the accumulation of endogenous and fluorescent-tagged proteins for DNA repair synthesis at the sites of DNA damage produced locally by UVA-laser micro-irradiation in HeLa cells. Accumulation kinetics and in vitro pull-down assays of the large subunit of RFC (RFC140) revealed that there are two distinct modes of recruitment of RFC to DNA damage, a simultaneous accumulation of RFC140 and PCNA caused by interaction between PCNA and the extreme N-terminus of RFC140 and a much faster accumulation of RFC140 than PCNA at the damaged site. Furthermore, RFC140 knock-down experiments showed that PCNA can accumulate at DNA damage independently of RFC. These results suggest that immediate accumulation of RFC and PCNA at DNA damage is only partly interdependent.     

Lys-110 is essential for targeting PCNA to replication and repair foci, and the K110A mutant activates apoptosis

Background information. PCNA (proliferating cell nuclear antigen) is required for a wide range of cellular functions, including DNA replication and damage repair. To be functional, PCNA must associate with the replication and repair foci. In addition, PCNA also mediates targeting of certain replication and repair proteins to these foci. However, the mechanism is not yet known by which PCNA is imported into the nucleus, and then localized to the replication and repair foci. Results. We have found that an NLS (nuclear localization sequence) is present within the amino acid 101–120 segment of PCNA. An NLS‐deleted PCNA was localized in the cytoplasm and showed 5‐fold lower affinity for importin‐β than wild‐type, suggesting that PCNA may be imported into the nucleus by importin‐β via its NLS. We previously reported that the functional unit of PCNA is a double trimer (as opposed to single homotrimer), and Lys‐110 is essential for the formation of the double trimer complex [Naryzhny, Zhao and Lee ( 2005 ) J. Biol. Chem. 280 , 13888–13894]. The present study shows that the substitution of Lys‐110 within the NLS to an alanine residue did not affect its nuclear localization. However, the double‐trimer‐defective PCNA(K110A) was not localized at replication or repair foci. In contrast, the double‐trimer‐intact PCNA(K117A) mutant was targeted normally to replication and repair foci. Interestingly, in cells transfected with PCNA(K110A), but not PCNA(K117A), caspase‐3‐mediated chromosome fragmentation was activated. Conclusions. The present study suggests that the regulation of PCNA is intimately connected with that of DNA replication, repair and cell death signals, and raises the possibility that defects in the formation of the PCNA double‐trimer complex can cause apoptosis.     

New Insights into Replication Clamp Unloading

The sliding clamp protein proliferating cell nuclear antigen (PCNA) is situated at the core of the eukaryotic replisome, where it acts as an interaction scaffold for numerous replication and repair factors and coordinates DNA transactions ranging from Okazaki fragment maturation to chromatin assembly and mismatch repair. PCNA is loaded onto DNA by a dedicated complex, the replication factor C, whose mechanism has been studied in detail. Until recently, however, it was unclear how PCNA is removed from DNA upon completion of DNA synthesis. Two complementary studies now present data strongly implicating the replication factor C-like complex, Elg1/ATAD5-RLC, in the unloading of PCNA during replication in yeast and human cells. They indicate that an appropriate control over PCNA's residence on the chromatin is important for maintaining genome stability. At the same time, they suggest that the interaction of Elg1/ATAD5 with SUMO, which was also reported to contribute to its role in genome maintenance, affects aspects of its function distinct from its unloading activity.     

The Pol30-K196 residue plays a critical role in budding yeast DNA postreplication repair through interaction with Rad18

PCNA plays critical roles in DNA replication and various DNA repair pathways including DNA damage tolerance (DDT). In budding yeast Saccharomyces cerevisiae, DDT (aka DNA postreplication repair, PRR) is achieved by sequential ubiquitination of PCNA encoded by POL30. Our previous studies revealed that two Arabidopsis PCNA genes were able to complement the essential function of POL30 in budding yeast, but failed to rescue the PRR activity. Here we hypothesize that a certain amino acid variation(s) is responsible for the difference, and identified K196 as a critical residue for the PRR activity. It was found that the pol30-K196V mutation abolishes Rad18 interaction and PRR activity, whereas nearby amino acid substitutions can partially restore Rad18 interaction and PRR activity. Together with the Pol30-Ub fusion data, we believe that we have identified a putative Rad18-binding pocket in Pol30 that is required for PCNA monoubiquitination and PRR.     

The C-terminal Domain of p21 Inhibits Nucleotide Excision Repair In Vitro and In Vivo

The protein p21(Cip1, Waf1, Sdi1) is a potent inhibitor of cyclin-dependent kinases (CDKs). p21 can also block DNA replication through its interaction with the proliferating cell nuclear antigen (PCNA), which is an auxiliary factor for polymerase delta. PCNA is also implicated in the repair resynthesis step of nucleotide excision repair (NER). Previous studies have yielded contradictory results on whether p21 regulates NER through its interaction with PCNA. Resolution of this controversy is of interest because it would help understand how DNA repair and replication are regulated. Hence, we have investigated the effect of p21 on NER both in vitro and in vivo using purified fragments of p21 containing either the CDK-binding domain (N terminus) or the PCNA binding domain (C terminus) of the protein. In the in vitro studies, DNA repair synthesis was measured in extracts from normal human fibroblasts using plasmids damaged by UV irradiation. In the in vivo studies, we used intact and permeabilized cells. The results show that the C terminus of the p21 protein inhibits NER both in vitro and in vivo. These are the first in vivo studies in which this question has been examined, and we demonstrate that inhibition of NER by p21 is not merely an artificial in vitro effect. A 50% inhibition of in vitro NER occurred at a 50:1 molar ratio of p21 C-terminus fragment to PCNA monomer. p21 differentially regulates DNA repair and replication, with repair being much less sensitive to inhibition than replication. Our in vivo results suggest that the inhibition occurs at the resynthesis step of the repair process. It also appears that preassembly of PCNA at repair sites mitigates the inhibitory effect of p21. We further demonstrate that the inhibition of DNA repair is mediated via binding of p21 to PCNA. The N terminus of p21 had no effect on DNA repair, and the inhibition of DNA repair by the C terminus of p21 was relieved by the addition of purified PCNA protein.     

DNA polymerase delta, RFC and PCNA are required for repair synthesis of large looped heteroduplexes in Saccharomyces cerevisiae

Small looped mispairs are corrected by DNA mismatch repair (MMR). In addition, a distinct process called large loop repair (LLR) corrects loops up to several hundred nucleotides in extracts of bacteria, yeast or human cells. Although LLR activity can be readily demonstrated, there has been little progress in identifying its protein components. This study identified some of the yeast proteins responsible for DNA repair synthesis during LLR. Polyclonal antisera to either Pol31 or Pol32 subunits of polymerase δ efficiently inhibited LLR in extracts by blocking repair just prior to gap filling. Gap filling was inhibited regardless of whether the loop was retained or removed. These experiments suggest polymerase δ is uniquely required in yeast extracts for LLR-associated synthesis. Similar results were obtained with antisera to the clamp loader proteins Rfc3 and Rfc4, and to PCNA, i.e. LLR was inhibited just prior to gap filling for both loop removal and loop retention. Thus PCNA and RFC seem to act in LLR only during repair synthesis, in contrast to their roles at both pre- and post-excision steps of MMR. These biochemical experiments support the idea that yeast polymerase δ, RFC and PCNA are required for large loop DNA repair synthesis.     

Cdt1 proteolysis is promoted by dual PIP degrons and is modulated by PCNA ubiquitylation

Cdt1 plays a critical role in DNA replication regulation by controlling licensing. In Metazoa, Cdt1 is regulated by CRL4(Cdt2)-mediated ubiquitylation, which is triggered by DNA binding of proliferating cell nuclear antigen (PCNA). We show here that fission yeast Cdt1 interacts with PCNA in vivo and that DNA loading of PCNA is needed for Cdt1 proteolysis after DNA damage and in S phase. Activation of this pathway by ultraviolet (UV)-induced DNA damage requires upstream involvement of nucleotide excision repair or UVDE repair enzymes. Unexpectedly, two non-canonical PCNA-interacting peptide (PIP) motifs, which both have basic residues downstream, function redundantly in Cdt1 proteolysis. Finally, we show that poly-ubiquitylation of PCNA, which occurs after DNA damage, reduces Cdt1 proteolysis. This provides a mechanism for fine-tuning the activity of the CRL4(Cdt2) pathway towards Cdt1, allowing Cdt1 proteolysis to be more efficient in S phase than after DNA damage.     

Distinct populations of human PCNA are required for initiation of chromosomal DNA replication and concurrent DNA repair

The integrity of genomic DNA during the cell division cycle in eukaryotic cells is maintained by regulated chromosomal DNA replication and repair of damaged DNA. We have used fractionation and reconstitution experiments to purify essential factors for the initiation of human chromosomal DNA replication in late G1 phase template nuclei from human cells. Here, we report the identification of soluble PCNA as an essential initiation factor in this system. Recombinant histidine-tagged human PCNA can substitute for purified endogenous human PCNA to initiate human chromosomal DNA replication. It is recruited specifically to discrete DNA replication foci formed during initiation in vitro. The template nuclei also contain DNA breaks as result of the synchronisation procedure. A separate population of chromatin-bound PCNA is already present in these template nuclei at discrete DNA damage foci, co-localising with gamma-H2AX, RPA and Rad51. This DNA damage-associated PCNA population is marked by mono-ubiquitination, suggesting that it is involved in DNA repair. Importantly, the population of damage focus-associated PCNA is neither involved in, nor required for, the initiation of chromosomal DNA replication in the same nuclei.     

Phosphorylation of human INO80 is involved in DNA damage tolerance

Double strand breaks (DSBs) are the most serious type of DNA damage. DSBs can be generated directly by exposure to ionizing radiation or indirectly by replication fork collapse. The DNA damage tolerance pathway, which is conserved from bacteria to humans, prevents this collapse by overcoming replication blockages. The INO80 chromatin remodeling complex plays an important role in the DNA damage response. The yeast INO80 complex participates in the DNA damage tolerance pathway. The mechanisms regulating yINO80 complex are not fully understood, but yeast INO80 complex are necessary for efficient proliferating cell nuclear antigen (PCNA) ubiquitination and for recruitment of Rad18 to replication forks. In contrast, the function of the mammalian INO80 complex in DNA damage tolerance is less clear. Here, we show that human INO80 was necessary for PCNA ubiquitination and recruitment of Rad18 to DNA damage sites. Moreover, the C-terminal region of human INO80 was phosphorylated, and overexpression of a phosphorylation-deficient mutant of human INO80 resulted in decreased ubiquitination of PCNA during DNA replication. These results suggest that the human INO80 complex, like the yeast complex, was involved in the DNA damage tolerance pathway and that phosphorylation of human INO80 was involved in the DNA damage tolerance pathway. These findings provide new insights into the DNA damage tolerance pathway in mammalian cells.     

Postreplicative mismatch repair factors are recruited to Epstein-Barr virus replication compartments

The mismatch repair (MMR) system, highly conserved throughout evolution, corrects nucleotide mispairing that arise during cellular DNA replication. We report here that proliferating cell nuclear antigen (PCNA), the clamp loader complex (RF-C), and a series of MMR proteins like MSH-2, MSH-6, MLH1, and hPSM2 can be assembled to Epstein-Barr virus replication compartments, the sites of viral DNA synthesis. Levels of the DNA-bound form of PCNA increased with progression of viral productive replication. Bromodeoxyuridine-labeled chromatin immunodepletion analyses confirmed that PCNA is loaded onto newly synthesized viral DNA as well as BALF2 and BMRF1 viral proteins during lytic replication. Furthermore, the anti-PCNA, -MSH2, -MSH3, or -MSH6 antibodies could immunoprecipitate BMRF1 replication protein probably via the viral DNA genome. PCNA loading might trigger transfer of a series of host MMR proteins to the sites of viral DNA synthesis. The MMR factors might function for the repair of mismatches that arise during viral replication or act to inhibit recombination between moderately divergent (homologous) sequences.