PCNA Interaction Research Articles

PCNA recruits cohesin loader Scc2 to ensure sister chromatid cohesion

Sister chromatid cohesion, established during replication by the ring-shaped multiprotein complex cohesin, is essential for faithful chromosome segregation. Replisome-associated proteins are required to generate cohesion by two independent pathways. One mediates conversion of cohesins bound to unreplicated DNA ahead of replication forks into cohesive entities behind them, while the second promotes cohesin de novo loading onto newly replicated DNA. The latter process depends on the cohesin loader Scc2 (NIPBL in vertebrates) and the alternative PCNA loader CTF18-RFC. However, the mechanism of de novo cohesin loading during replication is unknown. Here we show that PCNA physically recruits the yeast cohesin loader Scc2 via its C-terminal PCNA-interacting protein motif. Binding to PCNA is crucial, as the scc2-pip mutant deficient in Scc2–PCNA interaction is defective in cohesion when combined with replisome mutants of the cohesin conversion pathway. Importantly, the role of NIPBL recruitment to PCNA for cohesion generation is conserved in vertebrate cells.

Ubiquitin specific peptidase 37 and PCNA interaction promotes osteosarcoma pathogenesis by modulating replication fork progression

BackgroundOsteosarcoma is a type of bone cancer that predominantly affects young individuals, including children and adolescents. The disease progresses through heterogeneous genetic alterations, and patients often develop pulmonary metastases even after the primary tumors have been surgically removed. Ubiquitin-specific peptidases (USPs) regulate several critical cellular processes, such as cell cycle progression, transcriptional activation, and signal transduction. Various studies have revealed the significance of USP37 in the regulation of replication stress and oncogenesis.MethodsIn this study, the Cancer Genome Atlas (TCGA) database was analyzed to investigate USP37 expression. RNA sequencing was utilized to assess the impact of USP37 overexpression and depletion on gene expression in osteosarcoma cells. Various molecular assays, including colony formation, immunofluorescence, immunoprecipitation, and DNA replication restart, were employed to examine the physical interaction between USP37 and PCNA, as well as its physiological effects in osteosarcoma cells. Additionally, molecular docking studies were conducted to gain insight into the nature of the interaction between USP37 and PCNA. Furthermore, immunohistochemistry was performed on archived tissue blocks from osteosarcoma patients to establish a correlation between USP37 and PCNA expression.ResultsAnalysis of the TCGA database revealed that increased expression of USP37 was linked to decreased progression-free survival (PFS) in osteosarcoma patients. Next-generation sequencing analysis of osteosarcoma cells demonstrated that overexpression or knockdown of USP37 led to the expression of different sets of genes. USP37 overexpression provided a survival advantage, while its depletion heightened sensitivity to replication stress in osteosarcoma cells. USP37 was found to physically interact with PCNA, and molecular docking studies indicated that the interaction occurs through unique residues. In response to genotoxic stress, cells that overexpressed USP37 resolved DNA damage foci more quickly than control cells or cells in which USP37 was depleted. The expression of USP37 varied in archived osteosarcoma tissues, with intermediate expression seen in 52% of cases in the cohort examined.ConclusionThe results of this investigation propose that USP37 plays a vital role in promoting replication stress tolerance in osteosarcoma cells. The interaction between USP37 and PCNA is involved in the regulation of replication stress, and disrupting it could potentially trigger synthetic lethality in osteosarcoma. This study has expanded our knowledge of the mechanism through which USP37 regulates replication stress, and its potential as a therapeutic target in osteosarcoma merits additional exploration.

RFWD3 promotes ZRANB3 recruitment to regulate the remodeling of stalled replication forks.

Replication fork reversal is an important mechanism to protect the stability of stalled forks and thereby preserve genomic integrity. While multiple enzymes have been identified that can remodel forks, their regulation remains poorly understood. Here, we demonstrate that the ubiquitin ligase RFWD3, whose mutation causes Fanconi Anemia, promotes recruitment of the DNA translocase ZRANB3 to stalled replication forks and ubiquitinated sites of DNA damage. Using electron microscopy, we show that RFWD3 stimulates fork remodeling in a ZRANB3-epistatic manner. Fork reversal is known to promote nascent DNA degradation in BRCA2-deficient cells. Consistent with a role for RFWD3 in fork reversal, inactivation of RFWD3 in these cells rescues fork degradation and collapse, analogous to ZRANB3 inactivation. RFWD3 loss impairs ZRANB3 localization to spontaneous nuclear foci induced by inhibition of the PCNA deubiquitinase USP1. We demonstrate that RFWD3 promotes PCNA ubiquitination and interaction with ZRANB3, providing a mechanism for RFWD3-dependent recruitment of ZRANB3. Together, these results uncover a new role for RFWD3 in regulating ZRANB3-dependent fork remodeling.

Pol32, an accessory subunit of DNA polymerase delta, plays an essential role in genome stability and pathogenesis of Candida albicans

ABSTRACT Candida albicans is a pathobiont that inflicts serious bloodstream fungal infections in individuals with compromised immunity and gut dysbiosis. Genomic diversity in the form of copy number alteration, ploidy variation, and loss of heterozygosity as an adaptive mechanism to adverse environments is frequently observed in C. albicans. Such genomic variations also confer a varied degree of fungal virulence and drug resistance, yet the factors propelling these are not completely understood. DNA polymerase delta (Polδ) is an essential replicative DNA polymerase in the eukaryotic cell and is yet to be characterized in C. albicans. Therefore, this study was designed to gain insights into the role of Polδ, especially its non-essential subunit Pol32, in the genome plasticity and life cycle of C. albicans. PCNA, the DNA clamp, recruits Polδ to the replication fork for processive DNA replication. Unlike in Saccharomyces cerevisiae, the PCNA interaction protein (PIP) motif of CaPol32 is critical for Polδ’s activity during DNA replication. Our comparative genetic analyses and whole-genome sequencing of POL32 proficient and deficient C. albicans cells revealed a critical role of Pol32 in DNA replication, cell cycle progression, and genome stability as SNPs, indels, and repeat variations were largely accumulated in pol32 null strain. The loss of pol32 in C. albicans conferred cell wall deformity; Hsp90 mediated azoles resistance, biofilm development, and a complete attenuation of virulence in an animal model of systemic candidiasis. Thus, although Pol32 is dispensable for cell survival, its function is essential for C. albicans pathogenesis; and we discuss its translational implications in antifungal drugs and whole-cell vaccine development.

Measuring the contributions of helicases to break-induced replication.

Break-Induced Replication (BIR) is a homologous recombination (HR) pathway that differentiates itself from all other HR pathways by involving extensive DNA synthesis of up to hundreds of kilobases. This DNA synthesis occurs in G2/M arrested cells by a mechanism distinct from regular DNA replication. BIR initiates by strand invasion of a single end of a DNA double-strand break (DSB) followed by extensive D-loop migration. The main replicative helicase Mcm2-7 is dispensable for BIR, however, Pif1 helicase and its PCNA interaction domain are required. Pif1 helicase was shown to be important for extensive repair-specific DNA synthesis at DSB in budding and fission yeasts, flies, and human cells, implicating conservation of the mechanism. Additionally, Mph1 helicase negatively regulates BIR by unwinding migrating D-loops, and Srs2 promotes BIR by eliminating the toxic joint molecules. Here, we describe the methods that address the following questions in studying BIR: (i) how to distinguish enzymes needed specifically for BIR from enzymes needed for other HR mechanisms that require short patch DNA synthesis, (ii) what are the phenotypes expected for mutants deficient in extensive synthesis during BIR, (iii) how to follow extensive DNA synthesis during BIR? Methods are described using yeast model organism and wild-type cells are compared side-by-side with Pif1 deficient cells.

The ubiquitin-binding domain of DNA polymerase η directly binds to DNA clamp PCNA and regulates translesion DNA synthesis

DNA polymerase eta (Polη) is a unique translesion DNA synthesis (TLS) enzyme required for the error-free bypass of ultraviolet ray (UV)-induced cyclobutane pyrimidine dimers in DNA. Therefore, its deficiency confers cellular sensitivity to UV radiation and an increased rate of UV-induced mutagenesis. Polη possesses a ubiquitin-binding zinc finger (ubz) domain and a PCNA-interacting-protein (pip) motif in the carboxy-terminal region. The role of the Polη pip motif in PCNA interaction required for DNA polymerase recruitment to the stalled replication fork has been demonstrated in earlier studies; however, the function of the ubz domain remains divisive. As per the current notion, the ubz domain of Polη binds to the ubiquitin moiety of the ubiquitinated PCNA, but such interaction is found to be nonessential for Polη's function. In this study, through amino acid sequence alignments, we identify three classes of Polη among different species based on the presence or absence of pip motif or ubz domain and using comprehensive mutational analyses, we show that the ubz domain of Polη, which intrinsically lacks the pip motif directly binds to the interdomain connecting loop (IDCL) of PCNA and regulates Polη's TLS activity. We further propose two distinct modes of PCNA interaction mediated either by pip motif or ubz domain in various Polη homologs. When the pip motif or ubz domain of a given Polη binds to the IDCL of PCNA, such interaction becomes essential, whereas the binding of ubz domain to PCNA through ubiquitin is dispensable for Polη's function.

Coordinated Cut and Bypass: Replication of Interstrand Crosslink-Containing DNA.

DNA interstrand crosslinks (ICLs) are covalently bound DNA lesions, which are commonly induced by chemotherapeutic drugs, such as cisplatin and mitomycin C or endogenous byproducts of metabolic processes. This type of DNA lesion can block ongoing RNA transcription and DNA replication and thus cause genome instability and cancer. Several cellular defense mechanism, such as the Fanconi anemia pathway have developed to ensure accurate repair and DNA replication when ICLs are present. Various structure-specific nucleases and translesion synthesis (TLS) polymerases have come into focus in relation to ICL bypass. Current models propose that a structure-specific nuclease incision is needed to unhook the ICL from the replication fork, followed by the activity of a low-fidelity TLS polymerase enabling replication through the unhooked ICL adduct. This review focuses on how, in parallel with the Fanconi anemia pathway, PCNA interactions and ICL-induced PCNA ubiquitylation regulate the recruitment, substrate specificity, activity, and coordinated action of certain nucleases and TLS polymerases in the execution of stalled replication fork rescue via ICL bypass.

Frame-shift mediated reduction of gain-of-function p53 R273H and deletion of the R273H C-terminus in breast cancer cells result in replication-stress sensitivity

We recently documented that gain-of-function (GOF) mutant p53 (mtp53) R273H in triple negative breast cancer (TNBC) cells interacts with replicating DNA and PARP1. The missense R273H GOF mtp53 has a mutated central DNA binding domain that renders it unable to bind specifically to DNA, but maintains the capacity to interact tightly with chromatin. Both the C-terminal domain (CTD) and oligomerization domain (OD) of GOF mtp53 proteins are intact and it is unclear whether these regions of mtp53 are responsible for chromatin-based DNA replication activities. We generated MDA-MB-468 cells with CRISPR-Cas9 edited versions of the CTD and OD regions of mtp53 R273H. These included a frame-shift mtp53 R273Hfs387, which depleted mtp53 protein expression; mtp53 R273HΔ381-388, which had a small deletion within the CTD; and mtp53 R273HΔ347-393, which had both the OD and CTD regions truncated. The mtp53 R273HΔ347-393 existed exclusively as monomers and disrupted the chromatin interaction of mtp53 R273H. The CRISPR variants proliferated more slowly than the parental cells and mt53 R273Hfs387 showed the most extreme phenotype. We uncovered that after thymidine-induced G1/S synchronization, but not hydroxyurea or aphidicholin, R273Hfs387 cells displayed impairment of S-phase progression while both R273HΔ347-393 and R273HΔ381-388 displayed only moderate impairment. Moreover, reduced chromatin interaction of MCM2 and PCNA in mtp53 depleted R273Hfs387 cells post thymidine-synchronization revealed delayed kinetics of replisome assembly underscoring the slow S-phase progression. Taken together our findings show that the CTD and OD domains of mtp53 R273H play critical roles in mutant p53 GOF that pertain to processes associated with DNA replication.

'PIPs' in DNA polymerase: PCNA interaction affairs.

Interaction of PCNA with DNA polymerase is vital to efficient and processive DNA synthesis. PCNA being a homotrimeric ring possesses three hydrophobic pockets mostly involved in an interaction with its binding partners. PCNA interacting proteins contain a short sequence of eight amino acids, popularly coined as PIP motif, which snuggly fits into the hydrophobic pocket of PCNA to stabilize the interaction. In the last two decades, several PIP motifs have been mapped or predicted in eukaryotic DNA polymerases. In this review, we summarize our understandings of DNA polymerase-PCNA interaction, the function of such interaction during DNA synthesis, and emphasize the lacunae that persist. Because of the presence of multiple ligands in the replisome complex and due to many interaction sites in DNA polymerases, we also propose two modes of DNA polymerase positioning on PCNA required for DNA synthesis to rationalize the tool-belt model of DNA replication.

FAM111A protects replication forks from protein obstacles via its trypsin-like domain

Persistent protein obstacles on genomic DNA, such as DNA-protein crosslinks (DPCs) and tight nucleoprotein complexes, can block replication forks. DPCs can be removed by the proteolytic activities of the metalloprotease SPRTN or the proteasome in a replication-coupled manner; however, additional proteolytic mechanisms may exist to cope with the diversity of protein obstacles. Here, we show that FAM111A, a PCNA-interacting protein, plays an important role in mitigating the effect of protein obstacles on replication forks. This function of FAM111A requires an intact trypsin-like protease domain, the PCNA interaction, and the DNA-binding domain that is necessary for protease activity in vivo. FAM111A, but not SPRTN, protects replication forks from stalling at poly(ADP-ribose) polymerase 1 (PARP1)-DNA complexes trapped by PARP inhibitors, thereby promoting cell survival after drug treatment. Altogether, our findings reveal a role of FAM111A in overcoming protein obstacles to replication forks, shedding light on cellular responses to anti-cancer therapies.

Interaction of apoptosis and pluripotency related transcripts for developmental potential of ovine embryos produced in vitro at different oxygen concentrations

The present study in sheep model was to find out the interaction of apoptotic transcripts, that is, Bcl2, Bax, Casp3, PCNA and p53 and pluripotency related transcripts, that is, Sox2, Nanog and Oct4 in ovine embryos produced in vitro at different O2 concentrations (20% and 5% O2) to compare their developmental potential. Oxygen concentrations did not influence the maturation and cleavage rate but the percentage of morula and blastocysts was significantly more at 5% as compared to 20% O2. A significant upregulated expression of Bcl2 and PCNA genes and significantly downregulated expression of Casp3 and p53 were observed in the blastocysts at 5% than those at 20% O2. The expression of Bax was not influenced by the O2 concentration. Among the pluripotency related transcripts, the expression of Oct4 was significantly upregulated and the expression of Sox2 and Nanog was significantly downregulated in embryos at 5% than at 20% O2. The study concluded that the embryos produced in vitro at low O2 (5%) concentration regulate the expression of developmental genes related to apoptosis and pluripotency to improve the developmental potential of embryos as compared to high O2 (20%) concentration.

Exploring new potential role of DDB2 by host cell reactivation assay in human tumorigenic cells

BackgroundThe Host Cell Reactivation assay (HCR) allows studying the DNA repair capability in different types of human cells. This assay was carried out to assess the ability in removing UV-lesions from DNA, thus verifying NER efficiency. Previously we have shown that DDB2, a protein involved in the Global Genome Repair, interacts directly with PCNA and, in human cells, the loss of this interaction affects DNA repair machinery. In addition, a mutant form unable to interact with PCNA (DDB2PCNA-), has shown a reduced ability to interact with a UV-damaged DNA plasmid in vitro.MethodsIn this work, we have investigated whether DDB2 protein may influence the repair of a UV-damaged DNA plasmid into the cellular environment by applying the HCR method. To this end, human kidney 293 stable clones, expressing DDB2Wt or DDB2PCNA-, were co-transfected with pmRFP-N2 and UV-irradiated pEGFP-reported plasmids. Moreover, the co-localization between DDB2 proteins and different NER factors recruited at DNA damaged sites was analysed by immunofluorescence and confocal microscopy.ResultsThe results have shown that DDB2Wt recognize and repair the UV-induced lesions in plasmidic DNA transfected in the cells, whereas a delay in these processes were observed in the presence of DDB2PCNA-, as also confirmed by the different extent of co-localization of DDB2Wt and some NER proteins (such as XPG), vs the DDB2 mutant form.ConclusionThe HCR confirms itself as a very helpful approach to assess in the cellular context the effect of expressing mutant vs Wt NER proteins on the DNA damage response. Loss of interaction of DDB2 and PCNA affects negatively DNA repair efficiency.

Interactional similarities and differences in the protein complex of PCNA and DNA replication factor C between rice and Arabidopsis

BackgroundProliferating cell nuclear antigen (PCNA), a conserved trimeric ring complex, is loaded onto replication fork through a hetero-pentameric AAA+ ATPase complex termed replication factor C (RFC) to maintain genome stability. Although architectures of PCNA-RFC complex in yeast have been revealed, the functions of PCNA and protein-protein interactions of PCNA-RFC complex in higher plants are not very clear. Here, essential regions mediating interactions between PCNA and RFC subunits in Arabidopsis and rice were investigated via yeast-two-hybrid method and bimolecular fluorescence complementation techniques.ResultsWe observed that OsPCNA could interact with all OsRFC subunits, while protein-protein interactions only exist between Arabidopsis RFC2/3/4/5 and AtPCNA1/2. The truncated analyses indicated that the C-terminal of Arabidopsis RFC2/3/4/5 and rice RFC1/2 is essential for binding PCNA while the region of rice RFC3/4/5 mediating interaction with PCNA distributed both at the N- and C-terminal. On the other hand, we found that the C- and N-terminal of Arabidopsis and rice PCNA contribute equally to PCNA-PCNA interaction, and the interdomain connecting loop (IDCL) domain and C-terminal of PCNAs are indispensable for interacting RFC subunits.ConclusionsThese results indicated that Arabidopsis and rice PCNAs are highly conserved in sequence, structure and pattern of interacting with other PCNA monomer. Nevertheless, there are also significant differences between the Arabidopsis and rice RFC subunits in binding PCNA. Taken together, our results could be helpful for revealing the biological functions of plant RFC-PCNA complex.

A Mutation in Rad54 that Affects PCNA Interaction Results in Decreased Homologous Recombination Events Following Double‐Strand Breaks

The Rad54 protein has multiple roles in the double‐strand break repair pathway of homologous recombination (HR). When DNA is damaged and the repair mechanisms (HR) are hindered, the DNA can remain nonfunctional, or worse, lead to maladies such as cancer. It is essential to understand the genes regulating HR in order to treat these disorders. We looked into one such critical mechanism in this project. Rad54 interacts with PCNA, the eukaryotic DNA replication clamp, but the function of this interaction is unknown. We hypothesize that this interaction is important in the steps of HR that require DNA synthesis. To determine the function of this interaction in Saccharomyces cerevisiae, a series of point mutants of rad54 were made and one, K102A/R103A (rad54‐KR/AA), was found to be defective for this interaction. The PCNA interaction is about 5‐fold reduced in the rad54‐KR/AA mutant protein, as measured by microscale thermophoresis. In addition, this mutant protein was tested in vitro for ATPase activity and the ability to bind other recombination proteins, notably the Rad51 recombinase. The ATPase activity and Rad51 interaction are intact, suggesting it is a true separation‐of‐function mutation. This phenotypic impact of this PCNA interaction mutation is being examined in vivo using assays that measure HR at the mating type locus. Strand invasion and repair synthesis are being tested using a PCR‐based assay, still in progress. The ability to complete recombination and switch mating types is known to require Rad54, so we are testing the rad54‐KR/AA mutant by inducing breaks at the MAT locus, plating cells and determining the number of cells that have switched mating type. So far we have found that the percentage of rad54‐KR/AA cells that can switch mating type is considerably lower than that of the wild type. This indicates that the Rad54‐PCNA interaction is required to efficiently repair double strand breaks. The fact that the Rad54‐KR/AA mutant protein is still capable of performing the switch albeit at a less frequent rate indicates that while that protein region is important for interaction with PCNA and for repair, it is not essential‐ it does not cripple the entire mechanism. In the PCR‐based assay, we expect to see less HR synthesis product because switching is reduced. This would indicate that rad54 KR/AA mutant protein region plays an important part in PCNA interactions‐ which are vital for HR; these interactions being a critical step in the final steps of synthesis and ultimately repair. When we know more about how these three components (Rad54, Rad51, and PCNA) all work together to repair damaged DNA, it will be a critical development in the field of genetic repair. Once we know the physiology of this damage, we stand a better chance of finding improved treatments to homologous recombination disorders, and possibly even cancer as a whole.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Human DNA polymerase delta is a pentameric holoenzyme with a dimeric p12 subunit.

Human DNA polymerase delta (Polδ), a holoenzyme consisting of p125, p50, p68, and p12 subunits, plays an essential role in DNA replication, repair, and recombination. Herein, using multiple physicochemical and cellular approaches, we found that the p12 protein forms a dimer in solution. In vitro reconstitution and pull down of cellular Polδ by tagged p12 substantiate the pentameric nature of this critical holoenzyme. Furthermore, a consensus proliferating nuclear antigen (PCNA) interaction protein motif at the extreme carboxyl-terminal tail and a homodimerization domain at the amino terminus of the p12 subunit were identified. Mutational analyses of these motifs in p12 suggest that dimerization facilitates p12 binding to the interdomain connecting loop of PCNA. In addition, we observed that oligomerization of the smallest subunit of Polδ is evolutionarily conserved as Cdm1 of Schizosaccharomyces pombe also dimerizes. Thus, we suggest that human Polδ is a pentameric complex with a dimeric p12 subunit, and discuss implications of p12 dimerization in enzyme architecture and PCNA interaction during DNA replication.

The Main Role of Srs2 in DNA Repair Depends on Its Helicase Activity, Rather than on Its Interactions with PCNA or Rad51.

ABSTRACTHomologous recombination (HR) is a mechanism that repairs a variety of DNA lesions. Under certain circumstances, however, HR can generate intermediates that can interfere with other cellular processes such as DNA transcription or replication. Cells have therefore developed pathways that abolish undesirable HR intermediates. The Saccharomyces cerevisiae yeast Srs2 helicase has a major role in one of these pathways. Srs2 also works during DNA replication and interacts with the clamp PCNA. The relative importance of Srs2’s helicase activity, Rad51 removal function, and PCNA interaction in genome stability remains unclear. We created a new SRS2 allele [srs2(1-850)] that lacks the whole C terminus, containing the interaction site for Rad51 and PCNA and interactions with many other proteins. Thus, the new allele encodes an Srs2 protein bearing only the activity of the DNA helicase. We find that the interactions of Srs2 with Rad51 and PCNA are dispensable for the main role of Srs2 in the repair of DNA damage in vegetative cells and for proper completion of meiosis. On the other hand, it has been shown that in cells impaired for the DNA damage tolerance (DDT) pathways, Srs2 generates toxic intermediates that lead to DNA damage sensitivity; we show that this negative Srs2 activity requires the C terminus of Srs2. Dissection of the genetic interactions of the srs2(1-850) allele suggest a role for Srs2’s helicase activity in sister chromatid cohesion. Our results also indicate that Srs2’s function becomes more central in diploid cells.

PAF-Myc-Controlled Cell Stemness Is Required for Intestinal Regeneration and Tumorigenesis

The underlying mechanisms of how self-renewing cells are controlled in regenerating tissues and cancer remain ambiguous. PCNA-associated factor (PAF) modulates DNA repair via PCNA. Also, PAF hyperactivates Wnt/β-catenin signaling independently of PCNA interaction. We found that PAF is expressed in intestinal stem and progenitor cells (ISCs and IPCs) and markedly upregulated during intestinal regeneration and tumorigenesis. Whereas PAF is dispensable for intestinal homeostasis, upon radiation injury, genetic ablation of PAF impairs intestinal regeneration along with the severe loss of ISCs and Myc expression. Mechanistically, PAF conditionally occupies and transactivates the c-Myc promoter, which induces the expansion of ISCs/IPCs during intestinal regeneration. In mouse models, PAF knockout inhibits Apc inactivation-driven intestinal tumorigenesis with reduced tumor cell stemness and suppressed Wnt/β-catenin signaling activity, supported by transcriptome profiling. Collectively, our results unveil that the PAF-Myc signaling axis is indispensable for intestinal regeneration and tumorigenesis by positively regulating self-renewing cells.

Role of the Pif1-PCNA Complex in Pol δ-Dependent Strand Displacement DNA Synthesis and Break-Induced Replication

The S.cerevisiae Pif1 helicase functions with DNA polymerase (Pol) δ in DNA synthesis during break-induced replication (BIR), a conserved pathway responsible for replication fork repair and telomere recombination. Pif1 interacts with the DNA polymerase processivity clamp PCNA, but the functional significance of the Pif1-PCNA complex remains to be elucidated. Here, we solve the crystal structure of PCNA in complex with a non-canonical PCNA-interacting motif in Pif1. The structure guides the construction of a Pif1 mutant that is deficient in PCNA interaction. This mutation impairs the ability of Pif1 to enhance DNA strand displacement synthesis by Pol δ invitro and also the efficiency of BIR in cells. These results provide insights into the role of the Pif1-PCNA-Polδ ensemble during DNA break repair by homologous recombination.

Characterization of a coupled DNA replication and translesion synthesis polymerase supraholoenzyme from archaea.

The ability of the replisome to seamlessly coordinate both high fidelity and translesion DNA synthesis requires a means to regulate recruitment and binding of enzymes from solution. Co-occupancy of multiple DNA polymerases within the replisome has been observed primarily in bacteria and is regulated by posttranslational modifications in eukaryotes, and both cases are coordinated by the processivity clamp. Because of the heterotrimeric nature of the PCNA clamp in some archaea, there is potential to occupy and regulate specific polymerases at defined subunits. In addition to specific PCNA and polymerase interactions (PIP site), we have now identified and characterized a novel protein contact between the Y-family DNA polymerase and the B-family replication polymerase (YB site) bound to PCNA and DNA from Sulfolobus solfataricus. These YB contacts are essential in forming and stabilizing a supraholoenzyme (SHE) complex on DNA, effectively increasing processivity of DNA synthesis. The SHE complex can not only coordinate polymerase exchange within the complex but also provides a mechanism for recruitment of polymerases from solution based on multiequilibrium processes. Our results provide evidence for an archaeal PCNA ‘tool-belt’ recruitment model of multienzyme function that can facilitate both high fidelity and translesion synthesis within the replisome during DNA replication.

POLD3 Plays an Essential Role in Somatic Hypermutation

Abstract Somatic hypermutation (SHM) increases the affinity of antibodies to antigen. While the initial steps of SHM, including development of the lesion, its excision and formation of an abasic site, are well established, the repair and mutation events are still under investigation. Multiple DNA repair pathways have been suggested for the SHM. POLD3, as a part of a scaffold complex, connects error free or error prone DNA polymerases with the processivity factor PCNA and this interaction plays an essential role in mutagenesis and recombination. In this study we tested the role of POLD3 in SHM. We demonstrated elevated expression of POLD3 in activated mice B cells and accumulation of this protein at DNA damaged sites. We created a Burkett’s lymphoma Raji cell clone (Raji 2C9 clone) expressing endogenous mutant POLD3 lacking C-terminus PCNA interaction site, using the CRISPR system. Functional studies shown that the Raji cells expressing mutant variant demonstrated abnormal cell cycle distribution, hyper-sensitivity to genotoxic agents and compromised SHM, compare to the control cells. The frequency of mutations in locus encoding immunoglobulin heavy chain variable region was are five times lower in cells expressing mutant POLD3 compare to control cells. In control cells, mutations accumulated at the RGYW/WRCY “hot spots”, and majority of mutations were C to T or G to A transitions. In cells expressing mutant POLD3 mutations accumulated outside “hot spots” and are represented by transversions. The overall results indicate that POLD3 plays an important role in SHM.