PCNA Unloading Research Articles

PCNA Unloading Is Crucial for the Bypass of DNA Lesions Using Homologous Recombination.

DNA Damage Tolerance (DDT) mechanisms allow cells to bypass lesions in the DNA during replication. This allows the cells to progress normally through the cell cycle in the face of abnormalities in their DNA. PCNA, a homotrimeric sliding clamp complex, plays a central role in the coordination of various processes during DNA replication, including the choice of mechanism used during DNA damage bypass. Mono-or poly-ubiquitination of PCNA facilitates an error-prone or an error-free bypass mechanism, respectively. In contrast, SUMOylation recruits the Srs2 helicase, which prevents local homologous recombination. The Elg1 RFC-like complex plays an important role in unloading PCNA from the chromatin. We analyze the interaction of mutations that destabilize PCNA with mutations in the Elg1 clamp unloader and the Srs2 helicase. Our results suggest that, in addition to its role as a coordinator of bypass mechanisms, the very presence of PCNA on the chromatin prevents homologous recombination, even in the absence of the Srs2 helicase. Thus, PCNA unloading seems to be a pre-requisite for recombinational repair.

Short-range end resection requires ATAD5-mediated PCNA unloading for faithful homologous recombination.

Homologous recombination (HR) requires bidirectional end resection initiated by a nick formed close to a DNA double-strand break (DSB), dysregulation favoring error-prone DNA end-joining pathways. Here we investigate the role of the ATAD5, a PCNA unloading protein, in short-range end resection, long-range resection not being affected by ATAD5 deficiency. Rapid PCNA loading onto DNA at DSB sites depends on the RFC PCNA loader complex and MRE11-RAD50-NBS1 nuclease complexes bound to CtIP. Based on our cytological analyses and on an in vitro system for short-range end resection, we propose that PCNA unloading by ATAD5 is required for the completion of short-range resection. Hampering PCNA unloading also leads to failure to remove the KU70/80 complex from the termini of DSBs hindering DNA repair synthesis and the completion of HR. In line with this model, ATAD5-depleted cells are defective for HR, show increased sensitivity to camptothecin, a drug forming protein-DNA adducts, and an augmented dependency on end-joining pathways. Our study highlights the importance of PCNA regulation at DSB for proper end resection and HR.

Control of telomere length in yeast by SUMOylated PCNA and the Elg1 PCNA unloader.

Telomeres cap and protect the linear eukaryotic chromosomes. Telomere length is determined by an equilibrium between positive and negative regulators of telomerase activity. A systematic screen for yeast mutants that affect telomere length maintenance in the yeast Saccharomyces cerevisiae revealed that mutations in any of ~500 genes affects telomere length. One of the genes that, when mutated, causes telomere elongation is ELG1, which encodes an unloader of PCNA, the processivity factor for replicative DNA polymerases. PCNA can undergo SUMOylation on two conserved residues, K164 and K127, or ubiquitination at lysine 164. These modifications have already been implicated in genome stability processes. We report that SUMOylated PCNA acts as a signal that positively regulates telomerase activity. We also uncovered physical interactions between Elg1 and the CST (Cdc13-Stn1-Ten) complex and addressed the mechanism by which Elg1 and Stn1 negatively regulates telomere elongation, coordinated by SUMO. We discuss these results with respect to how chromosomal replication and telomere elongation are coordinated.

Control of telomere length in yeast by SUMOylated PCNA and the Elg1 PCNA unloader

Telomeres cap and protect the linear eukaryotic chromosomes. Telomere length is determined by an equilibrium between positive and negative regulators of telomerase activity. A systematic screen for yeast mutants that affect telomere length maintenance in the yeast Saccharomyces cerevisiae revealed that mutations in any of ~500 genes affects telomere length. One of the genes that, when mutated, causes telomere elongation is ELG1, which encodes an unloader of PCNA, the processivity factor for replicative DNA polymerases. PCNA can undergo SUMOylation on two conserved residues, K164 and K127, or ubiquitination at lysine 164. These modifications have already been implicated in genome stability processes. We report that SUMOylated PCNA acts as a signal that positively regulates telomerase activity. We also uncovered physical interactions between Elg1 and the CST (Cdc13-Stn1-Ten) complex and addressed the mechanism by which Elg1 and Stn1 negatively regulates telomere elongation, coordinated by SUMO. We discuss these results with respect to how chromosomal replication and telomere elongation are coordinated.

Effects of Defective Unloading and Recycling of PCNA Revealed by the Analysis of ELG1 Mutants.

Timely and complete replication of the genome is essential for life. The PCNA ring plays an essential role in DNA replication and repair by contributing to the processivity of DNA polymerases and by recruiting proteins that act in DNA replication-associated processes. The ELG1 gene encodes a protein that works, together with the Rfc2-5 subunits (shared by the replication factor C complex), to unload PCNA from chromatin. While ELG1 is not essential for life, deletion of the gene has strong consequences for the stability of the genome, and elg1 mutants exhibit sensitivity to DNA damaging agents, defects in genomic silencing, high mutation rates, and other striking phenotypes. Here, we sought to understand whether all the roles attributed to Elg1 in genome stability maintenance are due to its effects on PCNA unloading, or whether they are due to additional functions of the protein. By using a battery of mutants that affect PCNA accumulation at various degrees, we show that all the phenotypes measured correlate with the amount of PCNA left at the chromatin. Our results thus demonstrate the importance of Elg1 and of PCNA unloading in promoting proper chromatin structure and in maintaining a stable genome.

Lagging strand gap suppression connects BRCA-mediated fork protection to nucleosome assembly through PCNA-dependent CAF-1 recycling

The inability to protect stalled replication forks from nucleolytic degradation drives genome instability and underlies chemosensitivity in BRCA-deficient tumors. An emerging hallmark of BRCA-deficiency is the inability to suppress replication-associated single-stranded DNA (ssDNA) gaps. Here, we report that lagging strand ssDNA gaps interfere with the ASF1-CAF-1 nucleosome assembly pathway, and drive fork degradation in BRCA-deficient cells. We show that CAF-1 function at replication forks is lost in BRCA-deficient cells, due to defects in its recycling during replication stress. This CAF-1 recycling defect is caused by lagging strand gaps which preclude PCNA unloading, causing sequestration of PCNA-CAF-1 complexes on chromatin. Importantly, correcting PCNA unloading defects in BRCA-deficient cells restores CAF-1-dependent fork stability. We further show that the activation of a HIRA-dependent compensatory histone deposition pathway restores fork stability to BRCA-deficient cells. We thus define lagging strand gap suppression and nucleosome assembly as critical enablers of BRCA-mediated fork stability.

Rad53 arrests leading and lagging strand DNA synthesis via distinct mechanisms in response to DNA replication stress.

DNA replication stress threatens ordinary DNA synthesis. The evolutionarily conserved DNA replication stress response pathway involves sensor kinase Mec1/ATR, adaptor protein Mrc1/Claspin, and effector kinase Rad53/Chk1, which spurs a host of changes to stabilize replication forks and maintain genome integrity. DNA replication forks consist of largely distinct sets of proteins at leading and lagging strands that function autonomously in DNA synthesis in vitro. In this article, we discuss eSPAN and BrdU-IP-ssSeq, strand-specific sequencing technologies that permit analysis of protein localization and DNA synthesis at individual strands in budding yeast. Using these approaches, we show that under replication stress Rad53 stalls DNA synthesis on both leading and lagging strands. On lagging strands, it stimulates PCNA unloading, and on leading strands, it attenuates the replication function of Mrc1-Tof1. We propose that in doing so, Rad53 couples leading and lagging strand DNA synthesis during replication stress, thereby preventing the emergence of harmful ssDNA.

ATAD5 restricts R-loop formation through PCNA unloading and RNA helicase maintenance at the replication fork.

R-loops are formed when replicative forks collide with the transcriptional machinery and can cause genomic instability. However, it is unclear how R-loops are regulated at transcription-replication conflict (TRC) sites and how replisome proteins are regulated to prevent R-loop formation or mediate R-loop tolerance. Here, we report that ATAD5, a PCNA unloader, plays dual functions to reduce R-loops both under normal and replication stress conditions. ATAD5 interacts with RNA helicases such as DDX1, DDX5, DDX21 and DHX9 and increases the abundance of these helicases at replication forks to facilitate R-loop resolution. Depletion of ATAD5 or ATAD5-interacting RNA helicases consistently increases R-loops during the S phase and reduces the replication rate, both of which are enhanced by replication stress. In addition to R-loop resolution, ATAD5 prevents the generation of new R-loops behind the replication forks by unloading PCNA which, otherwise, accumulates and persists on DNA, causing a collision with the transcription machinery. Depletion of ATAD5 reduces transcription rates due to PCNA accumulation. Consistent with the role of ATAD5 and RNA helicases in maintaining genomic integrity by regulating R-loops, the corresponding genes were mutated or downregulated in several human tumors.

How yeast cells deal with stalled replication forks.

DNA polymerases sometimes stall during DNA replication at sites where DNA is damaged, or upon encounter with proteins or secondary structures of DNA. When that happens, the polymerase clamp PCNA can become modified with a single ubiquitin moiety at lysine 164, opening DNA Damage Tolerance (DDT) mechanisms that either repair or bypass the lesions. An alternative repair mechanism is the salvage recombination (SR) pathway, which copies information from the sister chromatid. SUMOylation of PCNA at the same lysine, or at lysine 127, can recruit the Srs2 helicase, which negatively controls SR. Recently, we have dissected the relationship between SR and the DDT pathways, and showed that overexpression of either the PCNA unloader Elg1, or the Rad52 homologous recombination protein, can bypass the repression by Srs2. Our results shed light on the interactions between different DNA damage repair/bypass proteins, and underscore the importance of PCNA modifications in organizing the complex task of dealing with DNA damage during replication of the genetic material.

Access to PCNA by Srs2 and Elg1 Controls the Choice between Alternative Repair Pathways in Saccharomyces cerevisiae.

During DNA replication, stalling can occur when the replicative DNA polymerases encounter lesions or hard-to replicate regions. Under these circumstances, the processivity factor PCNA gets ubiquitylated at lysine 164, inducing the DNA damage tolerance (DDT) mechanisms that can bypass lesions encountered during DNA replication. PCNA can also be SUMOylated at the same residue or at lysine 127. Surprisingly, pol30-K164R mutants display a higher degree of sensitivity to DNA-damaging agents than pol30-KK127,164RR strains, unable to modify any of the lysines. Here, we show that in addition to translesion synthesis and strand-transfer DDT mechanisms, an alternative repair mechanism ("salvage recombination") that copies information from the sister chromatid is repressed by the recruitment of Srs2 to SUMOylated PCNA. Overexpression of Elg1, the PCNA unloader, or of the recombination protein Rad52 allows its activation. We dissect the genetic requirements for this pathway, as well as the interactions between Srs2 and Elg1.IMPORTANCE PCNA, the ring that encircles DNA maintaining the processivity of DNA polymerases, is modified by ubiquitin and SUMO. Whereas ubiquitin is required for bypassing lesions through the DNA damage tolerance (DDT) pathways, we show here that SUMOylation represses another pathway, salvage recombination. The Srs2 helicase is recruited to SUMOylated PCNA and prevents the salvage pathway from acting. The pathway can be induced by overexpressing the PCNA unloader Elg1, or the homologous recombination protein Rad52. Our results underscore the role of PCNA modifications in controlling the various bypass and DNA repair mechanisms.

Ubiquitinated-PCNA protects replication forks from DNA2-mediated degradation by regulating Okazaki fragment maturation and chromatin assembly

Upon genotoxic stress, PCNA ubiquitination allows for replication of damaged DNA by recruiting lesion-bypass DNA polymerases. However, PCNA is also ubiquitinated during normal S-phase progression. By employing 293T and RPE1 cells deficient in PCNA ubiquitination, generated through CRISPR/Cas9 gene editing, here, we show that this modification promotes cellular proliferation and suppression of genomic instability under normal growth conditions. Loss of PCNA-ubiquitination results in DNA2-dependent but MRE11-independent nucleolytic degradation of nascent DNA at stalled replication forks. This degradation is linked to defective gap-filling in the wake of the replication fork and incomplete Okazaki fragment maturation, which interferes with efficient PCNA unloading by ATAD5 and subsequent nucleosome deposition by CAF-1. Moreover, concomitant loss of PCNA-ubiquitination and the BRCA pathway results in increased nascent DNA degradation and PARP inhibitor sensitivity. In conclusion, we show that by ensuring efficient Okazaki fragment maturation, PCNA-ubiquitination protects fork integrity and promotes the resistance of BRCA-deficient cells to PARP-inhibitors.

ATAD5 promotes replication restart by regulating RAD51 and PCNA in response to replication stress

Maintaining stability of replication forks is important for genomic integrity. However, it is not clear how replisome proteins contribute to fork stability under replication stress. Here, we report that ATAD5, a PCNA unloader, plays multiple functions at stalled forks including promoting its restart. ATAD5 depletion increases genomic instability upon hydroxyurea treatment in cultured cells and mice. ATAD5 recruits RAD51 to stalled forks in an ATR kinase-dependent manner by hydroxyurea-enhanced protein-protein interactions and timely removes PCNA from stalled forks for RAD51 recruitment. Consistent with the role of RAD51 in fork regression, ATAD5 depletion inhibits slowdown of fork progression and native 5-bromo-2ʹ-deoxyuridine signal induced by hydroxyurea. Single-molecule FRET showed that PCNA itself acts as a mechanical barrier to fork regression. Consequently, DNA breaks required for fork restart are reduced by ATAD5 depletion. Collectively, our results suggest an important role of ATAD5 in maintaining genome integrity during replication stress.

A role for the yeast PCNA unloader Elg1 in eliciting the DNA damage checkpoint.

During cell proliferation, the genome is constantly threatened by cellular and external factors. When the DNA is damaged, or when its faithful duplication is delayed by DNA polymerase stalling, the cells induce a coordinated response termed the DNA damage response (DDR) or checkpoint. Elg1 forms an RFC-like complex in charge of unloading the DNA polymerase processively factor PCNA during DNA replication and DNA repair. Using checkpoint-inducible strains, a recently published paper (Sau et al. in mBio 10(3):e01159-19. https://doi.org/10.1128/mbio.01159-19, 2019) uncovered a role for Elg1 in eliciting the DNA damage checkpoint (DC), one of the branches of the DDR. The apical kinase Mec1/ATR phosphorylates Elg1, as well as the adaptor proteins Rad9/53BP1 and Dpb11/TopBP1, which are recruited to the site of DNA damage to amplify the checkpoint signal. In the absence of Elg1, Rad9 and Dpb11 are recruited but fail to be phosphorylated and the signal is therefore not amplified. Thus, Elg1 appears to coordinate DNA repair and the induction of the DNA damage checkpoint.

The PCNA unloader Elg1 promotes recombination at collapsed replication forks in fission yeast.

Protein-DNA complexes can impede DNA replication and cause replication fork collapse. Whilst it is known that homologous recombination is deployed in such instances to restart replication, it is unclear how a stalled fork transitions into a collapsed fork at which recombination proteins can load. Previously we established assays in Schizosaccharomyces pombe for studying recombination induced by replication fork collapse at the site-specific protein-DNA barrier RTS1 (Nguyen et al., 2015). Here, we provide evidence that efficient recruitment/retention of two key recombination proteins (Rad51 and Rad52) to RTS1 depends on unloading of the polymerase sliding clamp PCNA from DNA by Elg1. We also show that, in the absence of Elg1, reduced recombination is partially suppressed by deleting fbh1 or, to a lesser extent, srs2, which encode known anti-recombinogenic DNA helicases. These findings suggest that PCNA unloading by Elg1 is necessary to limit Fbh1 and Srs2 activity, and thereby enable recombination to proceed.

Identification of Elg1 interaction partners and effects on post-replication chromatin re-formation.

Elg1, the major subunit of a Replication Factor C-like complex, is critical to ensure genomic stability during DNA replication, and is implicated in controlling chromatin structure. We investigated the consequences of Elg1 loss for the dynamics of chromatin re-formation following DNA replication. Measurement of Okazaki fragment length and the micrococcal nuclease sensitivity of newly replicated DNA revealed a defect in nucleosome organization in the absence of Elg1. Using a proteomic approach to identify Elg1 binding partners, we discovered that Elg1 interacts with Rtt106, a histone chaperone implicated in replication-coupled nucleosome assembly that also regulates transcription. A central role for Elg1 is the unloading of PCNA from chromatin following DNA replication, so we examined the relative importance of Rtt106 and PCNA unloading for chromatin reassembly following DNA replication. We find that the major cause of the chromatin organization defects of an ELG1 mutant is PCNA retention on DNA following replication, with Rtt106-Elg1 interaction potentially playing a contributory role.

POLE3-POLE4 Is a Histone H3-H4 Chaperone that Maintains Chromatin Integrity during DNA Replication

SummaryMaintenance of epigenetic integrity relies on coordinated recycling and partitioning of parental histones and deposition of newly synthesized histones during DNA replication. This process depends upon a poorly characterized network of histone chaperones, remodelers, and binding proteins. Here we implicate the POLE3-POLE4 subcomplex of the leading-strand polymerase, Polε, in replication-coupled nucleosome assembly through its ability to selectively bind to histones H3-H4. Using hydrogen/deuterium exchange mass spectrometry and physical mapping, we define minimal domains necessary for interaction between POLE3-POLE4 and histones H3-H4. Biochemical analyses establish that POLE3-POLE4 is a histone chaperone that promotes tetrasome formation and DNA supercoiling in vitro. In cells, POLE3-POLE4 binds both newly synthesized and parental histones, and its depletion hinders helicase unwinding and chromatin PCNA unloading and compromises coordinated parental histone retention and new histone deposition. Collectively, our study reveals that POLE3-POLE4 possesses intrinsic H3-H4 chaperone activity, which facilitates faithful nucleosome dynamics at the replication fork.

Single Molecule Study of ATAD5-Induced Unloading of PCNA

Single Molecule Study of ATAD5-Induced Unloading of PCNA

A New Method, "Reverse Yeast Two-Hybrid Array" (RYTHA), Identifies Mutants that Dissociate the Physical Interaction Between Elg1 and Slx5.

The vast majority of processes within the cell are carried out by proteins working in conjunction. The Yeast Two-Hybrid (Y2H) methodology allows the detection of physical interactions between any two interacting proteins. Here, we describe a novel systematic genetic methodology, "Reverse Yeast Two-Hybrid Array" (RYTHA), that allows the identification of proteins required for modulating the physical interaction between two given proteins. Our assay starts with a yeast strain in which the physical interaction of interest can be detected by growth on media lacking histidine, in the context of the Y2H methodology. By combining the synthetic genetic array technology, we can systematically screen mutant libraries of the yeast Saccharomyces cerevisiae to identify trans-acting mutations that disrupt the physical interaction of interest. We apply this novel method in a screen for mutants that disrupt the interaction between the N-terminus of Elg1 and the Slx5 protein. Elg1 is part of an alternative replication factor C-like complex that unloads PCNA during DNA replication and repair. Slx5 forms, together with Slx8, a SUMO-targeted ubiquitin ligase (STUbL) believed to send proteins to degradation. Our results show that the interaction requires both the STUbL activity and the PCNA unloading by Elg1, and identify topoisomerase I DNA-protein cross-links as a major factor in separating the two activities. Thus, we demonstrate that RYTHA can be applied to gain insights about particular pathways in yeast, by uncovering the connection between the proteasomal ubiquitin-dependent degradation pathway, DNA replication, and repair machinery, which can be separated by the topoisomerase-mediated cross-links to DNA.

A structure-function analysis of the yeast Elg1 protein reveals the importance of PCNA unloading in genome stability maintenance.

The sliding clamp, PCNA, plays a central role in DNA replication and repair. In the moving replication fork, PCNA is present at the leading strand and at each of the Okazaki fragments that are formed on the lagging strand. PCNA enhances the processivity of the replicative polymerases and provides a landing platform for other proteins and enzymes. The loading of the clamp onto DNA is performed by the Replication Factor C (RFC) complex, whereas its unloading can be carried out by an RFC-like complex containing Elg1. Mutations in ELG1 lead to DNA damage sensitivity and genome instability. To characterize the role of Elg1 in maintaining genomic integrity, we used homology modeling to generate a number of site-specific mutations in ELG1 that exhibit different PCNA unloading capabilities. We show that the sensitivity to DNA damaging agents and hyper-recombination of these alleles correlate with their ability to unload PCNA from the chromatin. Our results indicate that retention of modified and unmodified PCNA on the chromatin causes genomic instability. We also show, using purified proteins, that the Elg1 complex inhibits DNA synthesis by unloading SUMOylated PCNA from the DNA. Additionally, we find that mutations in ELG1 suppress the sensitivity of rad5Δ mutants to DNA damage by allowing trans-lesion synthesis to take place. Taken together, the data indicate that the Elg1–RLC complex plays an important role in the maintenance of genomic stability by unloading PCNA from the chromatin.

MutSα maintains the mismatch repair capability by inhibiting PCNA unloading.

Eukaryotic mismatch repair (MMR) utilizes single-strand breaks as signals to target the strand to be repaired. DNA-bound PCNA is also presumed to direct MMR. The MMR capability must be limited to a post-replicative temporal window during which the signals are available. However, both identity of the signal(s) involved in the retention of this temporal window and the mechanism that maintains the MMR capability after DNA synthesis remain unclear. Using Xenopus egg extracts, we discovered a mechanism that ensures long-term retention of the MMR capability. We show that DNA-bound PCNA induces strand-specific MMR in the absence of strand discontinuities. Strikingly, MutSα inhibited PCNA unloading through its PCNA-interacting motif, thereby extending significantly the temporal window permissive to strand-specific MMR. Our data identify DNA-bound PCNA as the signal that enables strand discrimination after the disappearance of strand discontinuities, and uncover a novel role of MutSα in the retention of the post-replicative MMR capability.