A novel heterotetrameric structure of the crenarchaeal PCNA2-PCNA3 complex

Proliferating cell nuclear antigen (PCNA) is a key protein that orchestrates the arrangement of DNA-processing proteins on DNA during DNA metabolism. In crenarchaea, PCNA forms a heterotrimer (PCNA123) consisting of PCNA1, PCNA2, and PCNA3, while in most eukaryotes and many archaea PCNAs form a homotrimer. Interestingly, unique oligomeric PCNAs from Sulfolobus tokodaii were reported in which PCNA2 and PCNA3 form a heterotrimer without PCNA1. In this paper, we describe the crystal structure of the stoPCNA2-stoPCNA3 complex. While most DNA sliding clamps form ring-shaped structures, our crystal structure showed an elliptic ring-like heterotetrameric complex, differing from a previous reports. Furthermore, we investigated the composition and the dimension of the stoPCNA2-stoPCNA3 complex in the solution using gel-filtration column chromatography and small-angle X-ray scattering analyses, respectively. These results indicate that stoPCNA2 and stoPCNA3 form the heterotetramer in solution. Based on our heterotetrameric structure, we propose a possible biological role for the heterotetrameric complex as a Holliday junction clamp.     

Three Proliferating Cell Nuclear Antigen-Like Proteins Found in the Hyperthermophilic Archaeon Aeropyrum pernix: Interactions with the Two DNA Polymerases

Proliferating cell nuclear antigen (PCNA) is an essential component in the eukaryotic DNA replication machinery, in which it works for tethering DNA polymerases on the DNA template to accomplish processive DNA synthesis. The PCNA also interacts with many other proteins in important cellular processes, including cell cycle control, DNA repair, and an apoptotic pathway in the domain EUCARYA: We identified three genes encoding PCNA-like sequences in the genome of Aeropyrum pernix, a crenarchaeal archaeon. We cloned and expressed these genes in Escherichia coli and analyzed the gene products. All three PCNA homologs stimulated the primer extension activities of the two DNA polymerases, polymerase I (Pol I) and Pol II, identified in A. pernix to various extents, among which A. pernix PCNA 3 (ApePCNA3) provided a most remarkable effect on both Pol I and Pol II. The three proteins were confirmed to exist in the A. pernix cells. These results suggest that the three PCNAs work as the processivity factor of DNA polymerases in A. pernix cells under different conditions. In Eucarya, three checkpoint proteins, Hus1, Rad1, and Rad9, have been proposed to form a PCNA-like ring structure and may work as a sliding clamp for the translesion DNA polymerases. Therefore, it is very interesting that three active PCNAs were found in one archaeal cell. Further analyses are necessary to determine whether each PCNA has specific roles, and moreover, how they reveal different functions in the cells.     

Monomeric yeast PCNA mutants are defective in interacting with and stimulating the ATPase activity of RFC

Yeast PCNA is a homo-trimeric, ring-shaped DNA polymerase accessory protein that can encircle duplex DNA. The integrity of this multimeric sliding DNA clamp is maintained through the protein-protein interactions at the interfaces of adjacent subunits. To investigate the importance of trimer stability for PCNA function, we introduced single amino acid substitutions at residues (A112T, S135F) that map to opposite ends of the monomeric protein. Recombinant wild-type and mutant PCNAs were purified from E. coli, and they were tested for their properties in vitro. Unlike the stable wild-type PCNA trimers, the mutant PCNA proteins behaved as monomers when diluted to low nanomolar concentrations. In contrast to what has been reported for a monomeric form of the beta clamp in E. coli, the monomeric PCNAs were compromised in their ability to interact with their associated clamp loader, replication factor C (RFC). Similarly, monomeric PCNAs were not effective in stimulating the ATPase activity of RFC. The mutant PCNAs were able to form mixed trimers with wild-type subunits, although these mixed trimers were unstable when loaded onto DNA. They were able to function as weak DNA polymerase delta processivity factors in vitro, and when the monomeric PCNA-41 (A112T, S135F double mutant) allele was introduced as the sole source of PCNA in vivo, the cells were viable and healthy. These pol30-41 mutants were, however, sensitive to UV irradiation and to the DNA damaging agent methylmethane sulfonate, implying that DNA repair pathways have a distinct requirement for stable DNA clamps.     

Proliferating cell nuclear antigen (PCNA): a key factor in DNA replication and cell cycle regulation

Background

PCNA (proliferating cell nuclear antigen) has been found in the nuclei of yeast, plant and animal cells that undergo cell division, suggesting a function in cell cycle regulation and/or DNA replication. It subsequently became clear that PCNA also played a role in other processes involving the cell genome.

Scope

This review discusses eukaryotic PCNA, with an emphasis on plant PCNA, in terms of the protein structure and its biochemical properties as well as gene structure, organization, expression and function. PCNA exerts a tripartite function by operating as (1) a sliding clamp during DNA synthesis, (2) a polymerase switch factor and (3) a recruitment factor. Most of its functions are mediated by its interactions with various proteins involved in DNA synthesis, repair and recombination as well as in regulation of the cell cycle and chromatid cohesion. Moreover, post-translational modifications of PCNA play a key role in regulation of its functions. Finally, a phylogenetic comparison of PCNA genes suggests that the multi-functionality observed in most species is a product of evolution.

Conclusions

Most plant PCNAs exhibit features similar to those found for PCNAs of other eukaryotes. Similarities include: (1) a trimeric ring structure of the PCNA sliding clamp, (2) the involvement of PCNA in DNA replication and repair, (3) the ability to stimulate the activity of DNA polymerase δ and (4) the ability to interact with p21, a regulator of the cell cycle. However, many plant genomes seem to contain the second, probably functional, copy of the PCNA gene, in contrast to PCNA pseudogenes that are found in mammalian genomes.     

A flexible interface between DNA ligase and PCNA supports conformational switching and efficient ligation of DNA

DNA sliding clamps encircle DNA and provide binding sites for many DNA-processing enzymes. However, it is largely unknown how sliding clamps like proliferating cell nuclear antigen (PCNA) coordinate multistep DNA transactions. We have determined structures of Sulfolobus solfataricus DNA ligase and heterotrimeric PCNA separately by X-ray diffraction and in complex by small-angle X-ray scattering (SAXS). Three distinct PCNA subunits assemble into a protein ring resembling the homotrimeric PCNA of humans but with three unique protein-binding sites. In the absence of nicked DNA, the Sulfolobus solfataricus DNA ligase has an open, extended conformation. When complexed with heterotrimeric PCNA, the DNA ligase binds to the PCNA3 subunit and ligase retains an open, extended conformation. A closed, ring-shaped conformation of ligase catalyzes a DNA end-joining reaction that is strongly stimulated by PCNA. This open-to-closed switch in the conformation of DNA ligase is accommodated by a malleable interface with PCNA that serves as an efficient platform for DNA ligation.     

A novel proteomic approach identifies new interaction partners for proliferating cell nuclear antigen

During DNA replication and repair, many proteins bind to and dissociate in a highly specific and ordered manner from proliferating cell nuclear antigen (PCNA). We describe a combined approach of in silico searches at the genome level and combinatorial peptide synthesis to investigate the binding properties of hundreds of short PCNA-interacting peptides (PIP-peptides) to archaeal and eukaryal PCNAs. Biological relevance of our combined approach was demonstrated by identification an inactive complex of Pyrococcus abyssi ribonuclease HII with PCNA. Furthermore we show that PIP-peptides interact with PCNA largely in a sequence independent manner. Our experimental approach also identified many so far unidentified PCNA interacting peptides in a number of human proteins.     

Human-Saccharomyces cerevisiae proliferating cell nuclear antigen hybrids: oligomeric structure and functional characterization using in vitro DNA replication

The proliferating cell nuclear antigen (PCNA) is a highly conserved protein required for the assembly of the DNA polymerase delta (pol delta) holoenzyme. Because PCNAs from Saccharomyces cerevisiae and human do not complement each other using in vitro or in vivo assays, hybrids of the two proteins would help identify region(s) involved in the assembly of the pol delta holoenzyme. Two mutants of human PCNA, HU1 (D21E) and HU3 (D120E), and six hybrids of human and S. cerevisiae PCNA, HC1, HC5, CH2, CH3, CH4, and CH5, were prepared by swapping corresponding regions between the two proteins. In solution, all PCNA assembled into trimers, albeit to different extents. These PCNA variants were tested for stimulation of pol delta and in vitro replication of M13 and SV40 DNA as well as to stimulate the ATPase activity of replication factor C (RF-C). Our data suggest that in addition to the interdomain connecting loop and C terminus, an additional site in the N terminus is required for pol delta interaction. PCNA mutants and hybrids that stimulated pol delta and RF-C were deficient in M13 and SV40 DNA replication assays, indicating that PCNA-induced pol delta stimulation and RF-C-mediated loading are not sufficient for coordinated DNA synthesis at a replication fork.     

Reduced stability and increased dynamics in the human proliferating cell nuclear antigen (PCNA) relative to the yeast homolog

Proliferating Cell Nuclear Antigen (PCNA) is an essential factor for DNA replication and repair. PCNA forms a toroidal, ring shaped structure of 90 kDa by the symmetric association of three identical monomers. The ring encircles the DNA and acts as a platform where polymerases and other proteins dock to carry out different DNA metabolic processes. The amino acid sequence of human PCNA is 35% identical to the yeast homolog, and the two proteins have the same 3D crystal structure. In this report, we give evidence that the budding yeast (sc) and human (h) PCNAs have highly similar structures in solution but differ substantially in their stability and dynamics. hPCNA is less resistant to chemical and thermal denaturation and displays lower cooperativity of unfolding as compared to scPCNA. Solvent exchange rates measurements show that the slowest exchanging backbone amides are at the β-sheet, in the structure core, and not at the helices, which line the central channel. However, all the backbone amides of hPCNA exchange fast, becoming undetectable within hours, while the signals from the core amides of scPCNA persist for longer times. The high dynamics of the α-helices, which face the DNA in the PCNA-loaded form, is likely to have functional implications for the sliding of the PCNA ring on the DNA since a large hole with a flexible wall facilitates the establishment of protein-DNA interactions that are transient and easily broken. The increased dynamics of hPCNA relative to scPCNA may allow it to acquire multiple induced conformations upon binding to its substrates enlarging its binding diversity.     

ATP-dependent interaction of human mismatch repair proteins and dual role of PCNA in mismatch repair.

DNA mismatch repair ensures genomic stability by correcting biosynthetic errors and by blocking homologous recombination. MutS-like and MutL-like proteins play important roles in these processes. In Escherichia coli and yeast these two types of proteins form a repair initiation complex that binds to mismatched DNA. However, whether human MutS and MutL homologs interact to form a complex has not been elucidated. Using immunoprecipitation and Western blot analysis we show here that human MSH2, MLH1, PMS2 and proliferating cell nuclear antigen (PCNA) can be co-immunoprecipitated, suggesting formation of a repair initiation complex among these proteins. Formation of the initiation complex is dependent on ATP hydrolysis and at least functional MSH2 and MLH1 proteins, because the complex could not be detected in tumor cells that produce truncated MLH1 or MSH2 protein. We also demonstrate that PCNA is required in human mismatch repair not only at the step of repair initiation, but also at the step of repair DNA re-synthesis.     

Interdomain connecting loop and J loop structures determine cross-species compatibility of PCNA

Eukaryotic proliferating cell nuclear antigen (PCNA) plays an essential role in orchestrating the assembly of the replisome complex, stimulating processive DNA synthesis, and recruiting other regulatory proteins during the DNA damage response. PCNA and its binding partner network are relatively conserved in eukaryotes, and it exhibits extraordinary structural similarity across species. However, despite this structural similarity, the PCNA of a given species is rarely functional in heterologous systems. In this report, we determined the X-ray crystal structure of Neurospora crassa PCNA (NcPCNA) and compared its structure–function relationship with other available PCNA studies to understand this cross-species incompatibility. We found two regions, the interdomain connecting loop (IDCL) and J loop structures, vary significantly among PCNAs. In particular, the J loop deviates in NcPCNA from that in Saccharomyces cerevisiae PCNA (ScPCNA) by 7 Å. Differences in the IDCL structures result in varied binding affinities of PCNAs for the subunit Pol32 of DNA polymerase delta and for T2-amino alcohol, a small-molecule inhibitor of human PCNA. To validate that these structural differences are accountable for functional incompatibility in S. cerevisiae, we generated NcPCNA mutants mimicking IDCL and J loop structures of ScPCNA. Our genetic analyses suggested that NcPCNA mutants are fully functional in S. cerevisiae. The susceptibility of the strains harboring ScPCNA mimics of NcPCNA to various genotoxic agents was similar to that in yeast cells expressing ScPCNA. Taken together, we conclude that in addition to the overall architecture of PCNA, structures of the IDCL and J loop of PCNA are critical determinants of interspecies functional compatibility.     

Crystal structures of the Arabidopsis thaliana proliferating cell nuclear antigen 1 and 2 proteins complexed with the human p21 C‐terminal segment

The proliferating cell nuclear antigen (PCNA) is well recognized as one of the essential cellular components of the DNA replication machinery in all eukaryotic organisms. Despite their prominent importance, very little biochemical and structural information about plant PCNAs is available, in comparison with that obtained from other eukaryotic organisms. We have determined the atomic resolution crystal structures of the two distinct Arabidopsis thaliana PCNAs (AtPCNA), both complexed with the C‐terminal segment of human p21. Both AtPCNAs form homotrimeric ring structures, which are essentially identical to each other, including the major contacts with the p21 peptide. The structure of the amino‐terminal half of the p21 peptide, containing the typical PIP box sequence, is remarkably similar to those observed in the previously reported crystal structures of the human and archaeal PCNA‐PIP box complexes. Meanwhile, the carboxy‐terminal halves of the p21 peptide in the plant PCNA complexes are bound to the protein in a unique manner, most probably because of crystal packing effects. A surface plasmon resonance analysis revealed high affinity between each AtPCNA and the C‐terminal fragment of human p21. This result strongly suggests that the interaction is functionally significant, although no plant homologs of p21 have been identified yet. We also discovered that AtPCNA1 and AtPCNA2 form heterotrimers, implying that hetero‐PCNA rings may play critical roles in cellular signal transduction, particularly in DNA repair.     

The human Rad9/Rad1/Hus1 damage sensor clamp interacts with DNA polymerase beta and increases its DNA substrate utilisation efficiency: implications for DNA repair

In eukaryotic cells, checkpoints are activated in response to DNA damage. This requires the action of DNA damage sensors such as the Rad family proteins. The three human proteins Rad9, Rad1 and Hus1 form a heterotrimeric complex (called the 9-1-1 complex) that is recruited onto DNA upon damage. DNA damage also triggers the recruitment of DNA repair proteins at the lesion, including specialized DNA polymerases. In this work, we showed that the 9-1-1 complex can physically interact with DNA polymerase β in vitro. Functional analysis revealed that the 9-1-1 complex had a stimulatory effect on DNA polymerase β activity. However, the presence of 9-1-1 complex neither affected DNA polymerase λ, another X family DNA polymerase, nor the two replicative DNA polymerases α and δ. DNA polymerase β stimulation resulted from an increase in its affinity for the primer–template and the interaction with the 9-1-1 complex stimulated deoxyribonucleotides misincorporation by DNA polymerase β. In addition, the 9-1-1 complex enhanced DNA strand displacement synthesis by DNA polymerase β on a 1 nt gap DNA substrate. Our data raise the possibility that the 9-1-1 complex might attract DNA polymerase β to DNA damage sites, thus connecting directly checkpoints and DNA repair.     

Characterization of the Replication Initiator Orc1/Cdc6 from the Archaeon Picrophilus torridus

Eukaryotic DNA replication is preceded by the assembly of prereplication complexes (pre-RCs) at or very near origins in G₁ phase, which licenses origin firing in S phase. The archaeal DNA replication machinery broadly resembles the eukaryal apparatus, though simpler in form. The eukaryotic replication initiator origin recognition complex (ORC), which serially recruits Cdc6 and other pre-RC proteins, comprises six components, Orc1-6. In archaea, a single gene encodes a protein similar to both the eukaryotic Cdc6 and the Orc1 subunit of the eukaryotic ORC, with most archaea possessing one to three Orc1/Cdc6 orthologs. Genome sequence analysis of the extreme acidophile Picrophilus torridus revealed a single Orc1/Cdc6 (PtOrc1/Cdc6). Biochemical analyses show MBP-tagged PtOrc1/Cdc6 to preferentially bind ORB (origin recognition box) sequences. The protein hydrolyzes ATP in a DNA-independent manner, though DNA inhibits MBP-PtOrc1/Cdc6-mediated ATP hydrolysis. PtOrc1/Cdc6 exists in stable complex with PCNA in Picrophilus extracts, and MBP-PtOrc1/Cdc6 interacts directly with PCNA through a PIP box near its C terminus. Furthermore, PCNA stimulates MBP-PtOrc1/Cdc6-mediated ATP hydrolysis in a DNA-dependent manner. This is the first study reporting a direct interaction between Orc1/Cdc6 and PCNA in archaea. The bacterial initiator DnaA is converted from an active to an inactive form by ATP hydrolysis, a process greatly facilitated by the bacterial ortholog of PCNA, the β subunit of Pol III. The stimulation of PtOrc1/Cdc6-mediated ATP hydrolysis by PCNA and the conservation of PCNA-interacting protein motifs in several archaeal PCNAs suggest the possibility of a similar mechanism of regulation existing in archaea. This mechanism may involve other yet to be identified archaeal proteins.     

Protein-protein interactions of yeast DNA polymerase III with mammalian and yeast proliferating cell nuclear antigen (PCNA)/cyclin

We have previously reported the purification of yeast analogs to mammalian DNA polymerase delta and proliferating-cell nuclear antigen (PCNA)/cyclin: DNA polymerase III and yeast PCNA, respectively. Through the use of gel-filtration chromatography, we have studied the interaction of the model template-primer system poly(dA).(dT)16 (40:1) with yeast DNA polymerase III and with PCNAs. Yeast DNA polymerase III binds to the DNA in the absence of yeast PCNA/cyclin, but comigration of either yeast or calf thymus PCNA/cyclin with the DNA requires the additional presence of yeast DNA polymerase III. We could also isolate a DNA-calf thymus DNA polymerase delta-calf thymus PCNA/cyclin complex. From these data, we propose that PCNA/cyclin is involved not in the binding step of the polymerase to the template-primer, but in the elongation step. The 3'----5' exonuclease associated with yeast DNA polymerase III acts in a distributive manner on poly(dA).(pT)16, and dissociates from the DNA when addition of dTTP allows switching from the exonuclease to the polymerase mode. Addition of PCNA/cyclin had no effect on these activities.     

Isolation and analysis of the fission yeast gene encoding polymerase delta accessory protein PCNA.

Five monoclonal antibodies raised against rat PCNA cross-reacted with a similar protein in the fission yeast Schizosaccharomyces pombe. One of these was used to screen an S.pombe cDNA expression library. An incomplete cDNA was isolated and used to screen a genomic library, identifying a single gene, designated pcn1+ (proliferating cell nuclear antigen). The gene encodes a protein of 260 amino acids, with a deduced sequence 52% identical to human and rat PCNAs, which are 98.5% identical to each other. The budding yeast PCNA homologue POL30 is only 35% identical to the human and rat proteins. Pcn1 has a region near the C-terminus of particularly high homology to higher eukaryotic PCNA proteins. pcn1+ is essential for viability and delta pcn1 cells undergo aberrant DNA replication before cell cycle arrest. Overproduction of the protein leads to cell cycle delay in G2. Disruption of pcn1+ is complemented by the human PCNA gene, demonstrating that these genes are functional homologues.     

9-1-1: PCNA's specialized cousin

All living organisms are vulnerable to DNA damage. Cells respond to this hazard by activating a complex network of checkpoint and repair proteins to preserve genomic integrity. The DNA-encircling, ring-shaped heterotrimeric 9-1-1 complex, a relative of the replication protein PCNA, is a central coordinator of these events. 9-1-1 is loaded to damaged sites where it serves as a platform for the selective recruitment of checkpoint and repair proteins. In this Opinion article, 9-1-1 and proliferating cell nuclear antigen (PCNA) are compared and discussed in light of their respective structures and functions. We propose that the interaction partners of 9-1-1 possess specific 9-1-1-interaction boxes, which discriminate between 9-1-1 and PCNA thereby enabling specific interactions with individual 9-1-1 subunits.     

Interaction between human mismatch repair recognition proteins and checkpoint sensor Rad9-Rad1-Hus1

In eukaryotic cells, the cell cycle checkpoint proteins Rad9, Rad1, and Hus1 form the 9-1-1 complex which is structurally similar to the proliferating cell nuclear antigen (PCNA) sliding clamp. hMSH2/hMSH6 (hMutSα) and hMSH2/hMSH3 (hMutSβ) are the mismatch recognition factors of the mismatch repair pathway. hMutSα has been shown to physically and functionally interact with PCNA. Moreover, DNA methylating agent N-methyl-N′-nitro-N-nitrosoguanidine (MNNG) treatment induces the G2/M cell cycle arrest that is dependent on the presence of hMutSα and hMutLα. In this study, we show that each subunit of the human 9-1-1 complex physically interacts with hMSH2, hMSH3, and hMSH6. The 9-1-1 complex from both humans and Schizosaccharomyces pombe can stimulate hMutSα binding with G/T-containing DNA. Rad9, Rad1, and Hus1 individual subunits can also stimulate the DNA binding activity of hMutSα. Human Rad9 and hMSH6 colocalize to nuclear foci of HeLa cells after exposure to MNNG. However, Rad9 does not form foci in MSH6 defective cells following MNNG treatment. In Rad9 knockdown untreated cells, the majority of the MSH6 is in cytoplasm. Following MNNG treatment, Rad9 knockdown cells has abnormal nuclear morphology and MSH6 is distributed around nuclear envelop. Our findings suggest that the 9-1-1 complex is a component of the mismatch repair involved in MNNG-induced damage response.     

Elg1, the major subunit of an alternative RFC complex,interacts with SUMO-processing proteins

PCNA is a homotrimeric ring with important roles in DNA replication and repair. PCNA is loaded and unloaded by the RFC complex, which is composed of five subunits (Rfc1-5). Three additional complexes that share with RFC the small subunits (Rfc2-5) and contain alternative large subunits were found in yeast and other eukaryotes. We have recently reported that one of these, the Elg1-RFC complex, interacts with SUMOylated PCNA and may play a role in its unloading during DNA repair. Here we report that a yeast-two-hybrid screen with the N terminus of Elg1(which interacts with SUMOylated PCNA) uncovered interactions with proteins that belong to the SUMO pathway, including Slx5 and Slx8, which form an E3 ubiquitin ligase that ubiquitinates SUMOylated proteins. Mutations in SLX5 result in a genomic instability phenotype similar to that of elg1 mutants. The physical interaction between the N terminus of Elg1 and Slx5 is mediated by poly-SUMO chains but not by PCNA modifications, and requires Siz2, but not Siz1, activity. Thus our results highlight the many important roles played by Elg1, some of which are PCNA-dependent and some PCNA-independent.     

Nuclear reorganization of DNA mismatch repair proteins in response to DNA damage

The DNA mismatch repair (MMR) system is highly conserved and vital for preserving genomic integrity. Current mechanistic models for MMR are mainly derived from in vitro assays including reconstitution of strand-specific MMR and DNA binding assays using short oligonucleotides. However, fundamental questions regarding the mechanism and regulation in the context of cellular DNA replication remain. Using synchronized populations of HeLa cells we demonstrated that hMSH2, hMLH1 and PCNA localize to the chromatin during S-phase, and accumulate to a greater extent in cells treated with a DNA alkylating agent. In addition, using small interfering RNA to deplete hMSH2, we demonstrated that hMLH1 localization to the chromatin is hMSH2-dependent. hMSH2/hMLH1/PCNA proteins, when associated with the chromatin, form a complex that is greatly enhanced by DNA damage. The DNA damage caused by high doses of alkylating agents leads to a G₂ arrest after only one round of replication. In these G₂-arrested cells, an hMSH2/hMLH1 complex persists on chromatin, however, PCNA is no longer in the complex. Cells treated with a lower dose of alkylating agent require two rounds of replication before cells arrest in G₂. In the first S-phase, the MMR proteins form a complex with PCNA, however, during the second S-phase PCNA is missing from that complex. The distinction between these complexes may suggest separate functions for the MMR proteins in damage repair and signaling. Additionally, using confocal immunofluorescence, we observed a population of hMSH6 that localized to the nucleolus. This population is significantly reduced after DNA damage suggesting that the protein is shuttled out of the nucleolus in response to damage. In contrast, hMLH1 is excluded from the nucleolus at all times. Thus, the nucleolus may act to segregate a population of hMSH2–hMSH6 from hMLH1–hPMS2 such that, in the absence of DNA damage, an inappropriate response is not invoked.     

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.