Characterization of human Spartan/C1orf124, an ubiquitin-PCNA interacting regulator of DNA damage tolerance

Unrepaired DNA damage may arrest ongoing replication forks, potentially resulting in fork collapse, increased mutagenesis and genomic instability. Replication through DNA lesions depends on mono- and polyubiquitylation of proliferating cell nuclear antigen (PCNA), which enable translesion synthesis (TLS) and template switching, respectively. A proper replication fork rescue is ensured by the dynamic ubiquitylation and deubiquitylation of PCNA; however, as yet, little is known about its regulation. Here, we show that human Spartan/C1orf124 protein provides a higher cellular level of ubiquitylated-PCNA by which it regulates the choice of DNA damage tolerance pathways. We find that Spartan is recruited to sites of replication stress, a process that depends on its PCNA- and ubiquitin-interacting domains and the RAD18 PCNA ubiquitin ligase. Preferential association of Spartan with ubiquitin-modified PCNA protects against PCNA deubiquitylation by ubiquitin-specific protease 1 and facilitates the access of a TLS polymerase to the replication fork. In concert, depletion of Spartan leads to increased sensitivity to DNA damaging agents and causes elevated levels of sister chromatid exchanges. We propose that Spartan promotes genomic stability by regulating the choice of rescue of stalled replication fork, whose mechanism includes its interaction with ubiquitin-conjugated PCNA and protection against PCNA deubiquitylation.     

New histone supply regulates replication fork speed and PCNA unloading

Correct duplication of DNA sequence and its organization into chromatin is central to genome function and stability. However, it remains unclear how cells coordinate DNA synthesis with provision of new histones for chromatin assembly to ensure chromosomal stability. In this paper, we show that replication fork speed is dependent on new histone supply and efficient nucleosome assembly. Inhibition of canonical histone biosynthesis impaired replication fork progression and reduced nucleosome occupancy on newly synthesized DNA. Replication forks initially remained stable without activation of conventional checkpoints, although prolonged histone deficiency generated DNA damage. PCNA accumulated on newly synthesized DNA in cells lacking new histones, possibly to maintain opportunity for CAF-1 recruitment and nucleosome assembly. Consistent with this, in vitro and in vivo analysis showed that PCNA unloading is delayed in the absence of nucleosome assembly. We propose that coupling of fork speed and PCNA unloading to nucleosome assembly provides a simple mechanism to adjust DNA replication and maintain chromatin integrity during transient histone shortage.     

Post-Translational Modifications of PCNA in Control of DNA Synthesis and DNA Damage Tolerance-the Implications in Carcinogenesis

The faithful DNA replication is a critical event for cell survival and inheritance. However, exogenous or endogenous sources of damage challenge the accurate synthesis of DNA, which causes DNA lesions. The DNA lesions are obstacles for replication fork progression. However, the prolonged replication fork stalling leads to replication fork collapse, which may cause DNA double-strand breaks (DSB). In order to maintain genomic stability, eukaryotic cells evolve translesion synthesis (TLS) and template switching (TS) to resolve the replication stalling. Proliferating cell nuclear antigen (PCNA) trimer acts as a slide clamp and encircles DNA to orchestrate DNA synthesis and DNA damage tolerance (DDT). The post-translational modifications (PTMs) of PCNA regulate these functions to ensure the appropriate initiation and termination of replication and DDT. The aberrant regulation of PCNA PTMs will result in DSB, which causes mutagenesis and poor response to chemotherapy. Here, we review the roles of the PCNA PTMs in DNA duplication and DDT. We propose that clarifying the regulation of PCNA PTMs may provide insights into understanding the development of cancers.     

Drosophila SUUR protein associates with PCNA and binds chromatin in a cell cycle-dependent manner

Drosophila SUUR (Suppressor of UnderReplication) protein was shown to regulate the DNA replication elongation process in endocycling cells. This protein is also known to be the component of silent chromatin in polyploid and diploid cells. To mark the different cell cycle stages, we used immunostaining patterns of PCNA, the main structural component of replication fork. We demonstrate that SUUR chromatin binding is dynamic throughout the endocyle in Drosophila salivary glands. We observed that SUUR chromosomal localization changed along with PCNA pattern and these proteins largely co-localized during the late S-phase in salivary glands. The hypothesized interaction between SUUR and PCNA was confirmed by co-immunoprecipitation from embryonic nuclear extracts. Our findings support the idea that the effect of SUUR on replication elongation depends on the cell cycle stage and can be mediated through its physical interaction with replication fork.     

Saccharomyces cerevisiae RRM3, a 5' to 3' DNA helicase,physically interacts with proliferating cell nuclear antigen

Proliferating cell nuclear antigen (PCNA) plays an essential role in eukaryotic DNA replication, and numerous DNA replication proteins have been found to interact with PCNA through a conserved eight-amino acid motif called the PIP-box. We have searched the genome of the yeast Saccharomyces cerevisiae for open reading frames that encode proteins with putative PIP-boxes and initiated testing of 135 novel candidates for their ability to interact with PCNA-conjugated agarose beads. The first new PCNA-binding protein identified in this manner is the 5' to 3' DNA helicase RRM3. Yeast two-hybrid tests show that N-terminal deletions of RRM3, which remove the PIP-box but leave the helicase motifs intact, abolish the interaction with PCNA. In addition, mutating the two phenylalanine residues in the PIP-box to alanine or aspartic acid reduces binding to PCNA, confirming that the PIP-box in RRM3 is responsible for interaction with PCNA. The results presented here suggest that the RRM3 helicase functions at the replication fork.     

PCNA ubiquitination and REV1 define temporally distinct mechanisms for controlling translesion synthesis in the avian cell line DT40

Translesion synthesis (TLS) is a potentially mutagenic method of bypassing DNA damage encountered during replication that requires the recruitment of specialized DNA polymerases to stalled replication forks or postreplicative gaps. Current models suggest that TLS is activated by monoubiquitination of the DNA sliding clamp PCNA. However, in higher organisms, fully effective TLS also requires a noncatalytic function of the Y family polymerase REV1. Using the genetically tractable chicken cell line DT40, we show that TLS at stalled replication forks requires that both the translesion polymerase-interaction domain and ubiquitin-binding domain in the C terminus of REV1 are intact. Surprisingly, however, PCNA ubiquitination is not required to maintain normal fork progression on damaged DNA. Conversely, PCNA ubiquitination is essential for filling postreplicative gaps. Thus, PCNA ubiquitination and REV1 play distinct roles in the coordination of DNA damage bypass that are temporally separated relative to replication fork arrest.     

Functional and physical interaction of yeast Mgs1 with PCNA: impact on RAD6-dependent DNA damage tolerance

Proliferating cell nuclear antigen (PCNA), a sliding clamp required for processive DNA synthesis, provides attachment sites for various other proteins that function in DNA replication, DNA repair, cell cycle progression and chromatin assembly. It has been shown that differential posttranslational modifications of PCNA by ubiquitin or SUMO play a pivotal role in controlling the choice of pathway for rescuing stalled replication forks. Here, we explored the roles of Mgs1 and PCNA in replication fork rescue. We provide evidence that Mgs1 physically associates with PCNA and that Mgs1 helps suppress the RAD6 DNA damage tolerance pathway in the absence of exogenous DNA damage. We also show that PCNA sumoylation inhibits the growth of mgs1 rad18 double mutants, in which PCNA sumoylation and the Srs2 DNA helicase coordinately prevent RAD52-dependent homologous recombination. The proposed roles for Mgs1, Srs2, and modified PCNA during replication arrest highlight the importance of modulating the RAD6 and RAD52 pathways to avoid genome instability.     

Identification of replication factor C from Saccharomyces cerevisiae: a component of the leading-strand DNA replication complex.

A number of proteins have been isolated from human cells on the basis of their ability to support DNA replication in vitro of the simian virus 40 (SV40) origin of DNA replication. One such protein, replication factor C (RFC), functions with the proliferating cell nuclear antigen (PCNA), replication protein A (RPA), and DNA polymerase delta to synthesize the leading strand at a replication fork. To determine whether these proteins perform similar roles during replication of DNA from origins in cellular chromosomes, we have begun to characterize functionally homologous proteins from the yeast Saccharomyces cerevisiae. RFC from S. cerevisiae was purified by its ability to stimulate yeast DNA polymerase delta on a primed single-stranded DNA template in the presence of yeast PCNA and RPA. Like its human-cell counterpart, RFC from S. cerevisiae (scRFC) has an associated DNA-activated ATPase activity as well as a primer-template, structure-specific DNA binding activity. By analogy with the phage T4 and SV40 DNA replication in vitro systems, the yeast RFC, PCNA, RPA, and DNA polymerase delta activities function together as a leading-strand DNA replication complex. Now that RFC from S. cerevisiae has been purified, all seven cellular factors previously shown to be required for SV40 DNA replication in vitro have been identified in S. cerevisiae.     

Interaction between PCNA and diubiquitinated Mcm10 is essential for cell growth in budding yeast

The minichromosome maintenance protein 10 (Mcm10) is an evolutionarily conserved factor that is essential for replication initiation and elongation. Mcm10 is part of the eukaryotic replication fork and interacts with a variety of proteins, including the Mcm2-7 helicase and DNA polymerase alpha/primase complexes. A motif search revealed a match to the proliferating cell nuclear antigen (PCNA)-interacting protein (PIP) box in Mcm10. Here, we demonstrate a direct interaction between Mcm10 and PCNA that is alleviated by mutations in conserved residues of the PIP box. Interestingly, only the diubiquitinated form of Mcm10 binds to PCNA. Diubiquitination of Mcm10 is cell cycle regulated; it first appears in late G(1) and persists throughout S phase. During this time, diubiquitinated Mcm10 is associated with chromatin, suggesting a direct role in DNA replication. Surprisingly, a Y245A substitution in the PIP box of Mcm10 that inhibits the interaction with PCNA abolishes cell proliferation. This severe-growth phenotype, which has not been observed for analogous mutations in other PCNA-interacting proteins, is rescued by a compensatory mutation in PCNA that restores interaction with Mcm10-Y245A. Taken together, our results suggest that diubiquitinated Mcm10 interacts with PCNA to facilitate an essential step in DNA elongation.     

Dynamic regulation of PCNA ubiquitylation/deubiquitylation

Proliferating Cell Nuclear Antigen (PCNA) ubiquitylation plays a crucial role in maintaining genomic stability during DNA replication. DNA damage stalling the DNA replication fork induces PCNA ubiquitylation that activates DNA damage bypass to prevent the collapse of DNA replication forks that could potentially produce double-strand breaks and chromosomal rearrangements. PCNA ubiquitylation dictates the mode of bypass depending on the level of ubiquitylation; monoubiquitylation and polyubiquitylation activate error-prone translesion synthesis and error-free template switching, respectively. Due to the error-prone nature of DNA damage bypass, PCNA ubiquitylation needs to be tightly regulated. Here, we review the molecular mechanisms to remove ubiquitin from PCNA including the emerging role of USP1 and ELG1 in this fascinating process.     

The Werner's Syndrome protein collaborates with REV1 to promote replication fork progression on damaged DNA

DNA damage tolerance pathways facilitate the bypass of DNA lesions encountered during replication. These pathways can be mechanistically divided into recombinational damage avoidance and translesion synthesis, in which the lesion is directly bypassed by specialised DNA polymerases. We have recently shown distinct genetic dependencies for lesion bypass at and behind the replication fork in the avian cell line DT40, bypass at the fork requiring REV1 and bypass at post-replicative gaps requiring PCNA ubiquitination by RAD18. The WRN helicase/exonuclease, which is mutated in the progeroid and cancer predisposition disorder Werner's Syndrome, has previously been implicated in a RAD18-dependent DNA damage tolerance pathway. However, WRN has also been shown to be required to maintain normal replication fork progression on a damaged DNA template, a defect reminiscent of REV1-deficient cells. Here we use the avian cell line DT40 to demonstrate that WRN assists REV1-dependent translesion synthesis at the replication fork and that PCNA ubiquitination-dependent post-replicative lesion bypass provides an important backup mechanism for damage tolerance in the absence of WRN protein.     

Polyubiquitinated PCNA Recruits the ZRANB3 Translocase to Maintain Genomic Integrity after Replication Stress

Completion of DNA replication after replication stress depends on PCNA, which undergoes monoubiquitination to stimulate direct bypass of DNA lesions by specialized DNA polymerases or is polyubiquitinated to promote recombination-dependent DNA synthesis across DNA lesions by template switching mechanisms. Here we report that the ZRANB3 translocase, a SNF2 family member related to the SIOD disorder SMARCAL1 protein, is recruited by polyubiquitinated PCNA to promote fork restart following replication arrest. ZRANB3 depletion in mammalian cells results in an increased frequency of sister chromatid exchange and DNA damage sensitivity after treatment with agents that cause replication stress. Using in?vitro biochemical assays, we show that recombinant ZRANB3 remodels DNA?structures mimicking stalled replication forks and disassembles recombination intermediates. We therefore propose that ZRANB3 maintains genomic stability at stalled or collapsed replication forks by facilitating fork restart and limiting inappropriate recombination that could occur during template switching events.     

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.     

Ubiquitylation of proliferating cell nuclear antigen and recruitment of human DNA polymerase eta

This study investigated the requirement for ubiquitylation of PCNA at lysine 164 during polymerase eta-dependent translesion synthesis (TLS) of site-specific cis-syn cyclobutane thymine dimers (T (wedge)T). The in vitro assay recapitulated origin-dependent initiation, fork assembly, and semiconservative, bidirectional replication of double-stranded circular DNA substrates. A phosphocellulose column was used to fractionate HeLa cell extracts into two fractions; flow-through column fraction I (CFI) contained endogenous PCNA, RPA, ubiquitin-activating enzyme E1, and ubiquitin conjugase Rad6, and eluted column fraction II (CFII) included pol delta, pol eta, and RFC. CFII supplemented with purified recombinant RPA and PCNA (wild type or K164R, in which lysine was replaced with arginine) was competent for DNA replication and TLS. K164R-PCNA complemented CFII for these activities to the same extent and efficiency as wild-type PCNA. CFII mixed with CFI (endogenous PCNA, E1, Rad6) exhibited enhanced DNA replication activity, but the same TLS efficiency determined with the purified proteins. These results demonstrate that PCNA ubiquitylation at K164 of PCNA is not required in vitro for pol eta to gain access to replication complexes at forks stalled by T (wedge)T and to catalyze TLS across this dimer.     

Post-replicative base excision repair in replication foci.

Base excision repair (BER) is initiated by a DNA glycosylase and is completed by alternative routes, one of which requires proliferating cell nuclear antigen (PCNA) and other proteins also involved in DNA replication. We report that the major nuclear uracil-DNA glycosylase (UNG2) increases in S phase, during which it co-localizes with incorporated BrdUrd in replication foci. Uracil is rapidly removed from replicatively incorporated dUMP residues in isolated nuclei. Neutralizing antibodies to UNG2 inhibit this removal, indicating that UNG2 is the major uracil-DNA glycosylase responsible. PCNA and replication protein A (RPA) co-localize with UNG2 in replication foci, and a direct molecular interaction of UNG2 with PCNA (one binding site) and RPA (two binding sites) was demonstrated using two-hybrid assays, a peptide SPOT assay and enzyme-linked immunosorbent assays. These results demonstrate rapid post-replicative removal of incorporated uracil by UNG2 and indicate the formation of a BER complex that contains UNG2, RPA and PCNA close to the replication fork.     

Role of SUMO modification of human PCNA at stalled replication fork

DNA double-strand breaks (DSBs) can be generated not only by reactive agents but also as a result of replication fork collapse at unrepaired DNA lesions. Whereas ubiquitylation of proliferating cell nuclear antigen (PCNA) facilitates damage bypass, modification of yeast PCNA by small ubiquitin-like modifier (SUMO) controls recombination by providing access for the Srs2 helicase to disrupt Rad51 nucleoprotein filaments. However, in human cells, the roles of PCNA SUMOylation have not been explored. Here, we characterize the modification of human PCNA by SUMO in vivo as well as in vitro. We establish that human PCNA can be SUMOylated at multiple sites including its highly conserved K164 residue and that SUMO modification is facilitated by replication factor C (RFC). We also show that expression of SUMOylation site PCNA mutants leads to increased DSB formation in the Rad18(-/-) cell line where the effect of Rad18-dependent K164 PCNA ubiquitylation can be ruled out. Moreover, expression of PCNA-SUMO1 fusion prevents DSB formation as well as inhibits recombination if replication stalls at DNA lesions. These findings suggest the importance of SUMO modification of human PCNA in preventing replication fork collapse to DSB and providing genome stability.     

Saccharomyces cerevisiae replication factor C. II. Formation and activity of complexes with the proliferating cell nuclear antigen and with DNA polymerases delta and epsilon.

Lag times in DNA synthesis by DNA polymerase delta holoenzyme were due to ATP-mediated formation of an initiation complex on the primed DNA by the polymerase with the proliferating cell nuclear antigen (PCNA) and replication factor C (RF-C). Lag time analysis showed that high affinity binding of RF-C to the primer terminus required PCNA and that this complex was recognized by the polymerase. The formation of stable complexes was investigated through their isolation by Bio-Gel A-5m filtration. A stable complex of RF-C and PCNA on primed single-stranded mp18 DNA was isolated when these factors were preincubated with the DNA and with ATP, or, less efficiently with ATP gamma S. These and additional experiments suggest that ATP binding promotes the formation of a labile complex of RF-C with PCNA at the primer terminus, whereas its hydrolysis is required to form a stable complex. Subsequently, DNA polymerase delta binds to either complex in a replication competent fashion without further energy requirement. DNA polymerase epsilon did not associate stably with RF-C and PCNA onto the DNA, but its transient participation with these cofactors into a holoenzyme-like initiation complex was inferred from its kinetic properties and replication product analysis. The kinetics of the elongation phase at 30 degrees, 110 nucleotides/s by DNA polymerase delta holoenzyme and 50 nucleotides/s by DNA polymerase epsilon holoenzyme, are in agreement with in vivo rates of replication fork movement in yeast. A model for the eukaryotic replication fork involving both DNA polymerase delta and epsilon is proposed.     

Spartan/C1orf124 is important to prevent UV-induced mutagenesis

Uninterrupted replication across damaged DNA is critical to prevent replication fork collapse and resulting double-strand DNA breaks. Rad18-mediated PCNA ubiquitination is a crucial event that triggers a number of downstream pathways important for lesion bypass. Here, we report characterization of Spartan, an evolutionarily conserved protein containing a PCNA-interacting peptide motif, called a PIP box, and a UBZ4 ubiquitin-binding domain. Spartan is a nuclear protein and forms DNA damage-induced foci that colocalize with markers for stalled DNA replication. Focus formation of Spartan requires its PIP-box and the UBZ4 domain and is dependent on Rad18 and the PCNA ubiquitination site, indicating that Spartan is recruited to ubiquitinated PCNA. Spartan depletion results in increased mutagenesis during replication of UV-damaged DNA. Taken together, our data suggest that Spartan is recruited to sites of stalled replication via ubiquitinated PCNA and plays an important role to prevent mutations associated with replication of damaged DNA.     

Spartan/C1orf124 is important to prevent UV-induced mutagenesis

Uninterrupted replication across damaged DNA is critical to prevent replication fork collapse and resulting double-strand DNA breaks. Rad18-mediated PCNA ubiquitination is a crucial event that triggers a number of downstream pathways important for lesion bypass. Here, we report characterization of Spartan, an evolutionarily conserved protein containing a PCNA-interacting peptide motif, called a PIP box, and a UBZ4 ubiquitin-binding domain. Spartan is a nuclear protein and forms DNA damage-induced foci that colocalize with markers for stalled DNA replication. Focus formation of Spartan requires its PIP-box and the UBZ4 domain and is dependent on Rad18 and the PCNA ubiquitination site, indicating that Spartan is recruited to ubiquitinated PCNA. Spartan depletion results in increased mutagenesis during replication of UV-damaged DNA. Taken together, our data suggest that Spartan is recruited to sites of stalled replication via ubiquitinated PCNA and plays an important role to prevent mutations associated with replication of damaged DNA.     

Unligated Okazaki Fragments Induce PCNA Ubiquitination and a Requirement for Rad59-Dependent Replication Fork Progression

Deficiency in DNA ligase I, encoded by CDC9 in budding yeast, leads to the accumulation of unligated Okazaki fragments and triggers PCNA ubiquitination at a non-canonical lysine residue. This signal is crucial to activate the S phase checkpoint, which promotes cell cycle delay. We report here that a pol30-K107 mutation alleviated cell cycle delay in cdc9 mutants, consistent with the idea that the modification of PCNA at K107 affects the rate of DNA synthesis at replication forks. To determine whether PCNA ubiquitination occurred in response to nicks or was triggered by the lack of PCNA-DNA ligase interaction, we complemented cdc9 cells with either wild-type DNA ligase I or a mutant form, which fails to interact with PCNA. Both enzymes reversed PCNA ubiquitination, arguing that the modification is likely an integral part of a novel nick-sensory mechanism and not due to non-specific secondary mutations that could have occurred spontaneously in cdc9 mutants. To further understand how cells cope with the accumulation of nicks during DNA replication, we utilized cdc9-1 in a genome-wide synthetic lethality screen, which identified RAD59 as a strong negative interactor. In comparison to cdc9 single mutants, cdc9 rad59Δ double mutants did not alter PCNA ubiquitination but enhanced phosphorylation of the mediator of the replication checkpoint, Mrc1. Since Mrc1 resides at the replication fork and is phosphorylated in response to fork stalling, these results indicate that Rad59 alleviates nick-induced replication fork slowdown. Thus, we propose that Rad59 promotes fork progression when Okazaki fragment processing is compromised and counteracts PCNA-K107 mediated cell cycle arrest.