SMARCAL1 ubiquitylation controls its association with RPA-coated ssDNA and promotes replication fork stability.

Impediments in replication fork progression cause genomic instability, mutagenesis, and severe pathologies. At stalled forks, RPA-coated single-stranded DNA (ssDNA) activates the ATR kinase and directs fork remodeling, 2 key early events of the replication stress response. RFWD3, a recently described Fanconi anemia (FA) ubiquitin ligase, associates with RPA and promotes its ubiquitylation, facilitating late steps of homologous recombination (HR). Intriguingly, RFWD3 also regulates fork progression, restart and stability via poorly understood mechanisms. Here, we used proteomics to identify putative RFWD3 substrates during replication stress in human cells. We show that RFWD3 interacts with and ubiquitylates the SMARCAL1 DNA translocase directly in vitro and following DNA damage in vivo. SMARCAL1 ubiquitylation does not trigger its subsequent proteasomal degradation but instead disengages it from RPA thereby regulating its function at replication forks. Proper regulation of SMARCAL1 by RFWD3 at stalled forks protects them from excessive MUS81-mediated cleavage in response to UV irradiation, thereby limiting DNA replication stress. Collectively, our results identify RFWD3-mediated SMARCAL1 ubiquitylation as a novel mechanism that modulates fork remodeling to avoid genome instability triggered by aberrant fork processing.

RPA interacts with Rad52 to promote meiotic crossover and noncrossover recombination.

Meiotic recombination is initiated by programmed double-strand breaks (DSBs). Studies in Saccharomyces cerevisiae have shown that, following rapid resection to generate 3' single-stranded DNA (ssDNA) tails, one DSB end engages a homolog partner chromatid and is extended by DNA synthesis, whereas the other end remains associated with its sister. Then, after regulated differentiation into crossover- and noncrossover-fated types, the second DSB end participates in the reaction by strand annealing with the extended first end, along both pathways. This second-end capture is dependent on Rad52, presumably via its known capacity to anneal two ssDNAs. Here, using physical analysis of DNA recombination, we demonstrate that this process is dependent on direct interaction of Rad52 with the ssDNA binding protein, replication protein A (RPA). Furthermore, the absence of this Rad52-RPA joint activity results in a cytologically-prominent RPA spike, which emerges from the homolog axes at sites of crossovers during the pachytene stage of the meiotic prophase. Our findings suggest that this spike represents the DSB end of a broken chromatid caused by either the displaced leading DSB end or the second DSB end, which has been unable to engage with the partner homolog-associated ssDNA. These and other results imply a close correspondence between Rad52-RPA roles in meiotic recombination and mitotic DSB repair.

Fluorescent human RPA to track assembly dynamics on DNA.

DNA metabolic processes including replication, repair, recombination, and telomere maintenance occur on single-stranded DNA (ssDNA). In each of these complex processes, dozens of proteins function together on the ssDNA template. However, when double-stranded DNA is unwound, the transiently open ssDNA is protected and coated by the high affinity heterotrimeric ssDNA binding Replication Protein A (RPA). Almost all downstream DNA processes must first remodel/remove RPA or function alongside to access the ssDNA occluded under RPA. Formation of RPA-ssDNA complexes trigger the DNA damage checkpoint response and is a key step in activating most DNA repair and recombination pathways. Thus, in addition to protecting the exposed ssDNA, RPA functions as a gatekeeper to define functional specificity in DNA maintenance and genomic integrity. RPA achieves functional dexterity through a multi-domain architecture utilizing several DNA binding and protein-interaction domains connected by flexible linkers. This flexible and modular architecture enables RPA to adopt a myriad of configurations tailored for specific DNA metabolic roles. To experimentally capture the dynamics of the domains of RPA upon binding to ssDNA and interacting proteins we here describe the generation of active site-specific fluorescent versions of human RPA (RPA) using 4-azido-L-phenylalanine (4AZP) incorporation and click chemistry. This approach can also be applied to site-specific modifications of other multi-domain proteins. Fluorescence-enhancement through non-canonical amino acids (FEncAA) and Förster Resonance Energy Transfer (FRET) assays for measuring dynamics of RPA on DNA are also described. The fluorescent human RPA described here will enable high-resolution structure-function analysis of RPA-ssDNA interactions.

Rapid Long-distance Migration of RPA on Single Stranded DNA Occurs Through Intersegmental Transfer Utilizing Multivalent Interactions.

Replication Protein A (RPA) is asingle strandedDNA(ssDNA)binding protein that coordinates diverse DNA metabolic processes including DNA replication, repair, and recombination. RPA is a heterotrimeric protein with six functional oligosaccharide/oligonucleotide (OB) domains and flexible linkers. Flexibility enables RPA to adopt multiple configurations andis thought to modulate its function. Here, usingsingle moleculeconfocal fluorescencemicroscopy combinedwith optical tweezers and coarse-grained molecular dynamics simulations, we investigated the diffusional migration of single RPA molecules on ssDNA undertension.The diffusioncoefficientDis the highest (20,000nucleotides2/s) at 3pNtension and in 100 mMKCl and markedly decreases whentensionor salt concentrationincreases. We attribute the tension effect to intersegmental transfer which is hindered by DNA stretching and the salt effect to an increase in binding site size and interaction energy of RPA-ssDNA. Our integrative study allowed us to estimate the size and frequency of intersegmental transfer events that occur through transient bridging of distant sites on DNA by multiple binding sites on RPA. Interestingly, deletion of RPA trimeric core still allowed significant ssDNA binding although the reduced contact area made RPA 15-fold more mobile. Finally, we characterized the effect of RPA crowding on RPA migration. These findings reveal how the high affinity RPA-ssDNA interactions are remodeled to yield access, a key step in several DNA metabolic processes.

Replication Protein A, the Main Eukaryotic Single-Stranded DNA Binding Protein, a Focal Point in Cellular DNA Metabolism.

Replication protein A (RPA) is a heterotrimeric protein complex and the main single-stranded DNA (ssDNA)-binding protein in eukaryotes. RPA has key functions in most of the DNA-associated metabolic pathways and DNA damage signalling. Its high affinity for ssDNA helps to stabilise ssDNA structures and protect the DNA sequence from nuclease attacks. RPA consists of multiple DNA-binding domains which are oligonucleotide/oligosaccharide-binding (OB)-folds that are responsible for DNA binding and interactions with proteins. These RPA-ssDNA and RPA-protein interactions are crucial for DNA replication, DNA repair, DNA damage signalling, and the conservation of the genetic information of cells. Proteins such as ATR use RPA to locate to regions of DNA damage for DNA damage signalling. The recruitment of nucleases and DNA exchange factors to sites of double-strand breaks are also an important RPA function to ensure effective DNA recombination to correct these DNA lesions. Due to its high affinity to ssDNA, RPA's removal from ssDNA is of central importance to allow these metabolic pathways to proceed, and processes to exchange RPA against downstream factors are established in all eukaryotes. These faceted and multi-layered functions of RPA as well as its role in a variety of human diseases will be discussed.

RPA guides UNG to uracil in ssDNA to facilitate antibody class switching and repair of mutagenic uracil at the replication fork.

Activation-induced cytidine deaminase (AID) interacts with replication protein A (RPA), the major ssDNA-binding protein, to promote deamination of cytosine to uracil in transcribed immunoglobulin (Ig) genes. Uracil-DNA glycosylase (UNG) acts in concert with AID during Ig diversification. In addition, UNG preserves genome integrity by base-excision repair (BER) in the overall genome. How UNG is regulated to support both mutagenic processing and error-free repair remains unknown. UNG is expressed as two isoforms, UNG1 and UNG2, which both contain an RPA-binding helix that facilitates uracil excision from RPA-coated ssDNA. However, the impact of this interaction in antibody diversification and genome maintenance has not been investigated. Here, we generated B-cell clones with targeted mutations in the UNG RPA-binding motif, and analysed class switch recombination (CSR), mutation frequency (5' Ig Sµ), and genomic uracil in clones representing seven Ung genotypes. We show that the UNG:RPA interaction plays a crucial role in both CSR and repair of AID-induced uracil at the Ig loci. By contrast, the interaction had no significant impact on total genomic uracil levels. Thus, RPA coordinates UNG during CSR and pre-replicative repair of mutagenic uracil in ssDNA but is not essential in post-replicative and canonical BER of uracil in dsDNA.

Human RAD52 stimulates the RAD51-mediated homology search.

Homologous recombination (HR) is a DNA repair mechanism of double-strand breaks and blocked replication forks, involving a process of homology search leading to the formation of synaptic intermediates that are regulated to ensure genome integrity. RAD51 recombinase plays a central role in this mechanism, supported by its RAD52 and BRCA2 partners. If the mediator function of BRCA2 to load RAD51 on RPA-ssDNA is well established, the role of RAD52 in HR is still far from understood. We used transmission electron microscopy combined with biochemistry to characterize the sequential participation of RPA, RAD52, and BRCA2 in the assembly of the RAD51 filament and its activity. Although our results confirm that RAD52 lacks a mediator activity, RAD52 can tightly bind to RPA-coated ssDNA, inhibit the mediator activity of BRCA2, and form shorter RAD51-RAD52 mixed filaments that are more efficient in the formation of synaptic complexes and D-loops, resulting in more frequent multi-invasions as well. We confirm the in situ interaction between RAD51 and RAD52 after double-strand break induction in vivo. This study provides new molecular insights into the formation and regulation of presynaptic and synaptic intermediates by BRCA2 and RAD52 during human HR.

Targeting RPA promotes autophagic flux and the antitumor response to radiation in nasopharyngeal carcinoma.

BACKGROUND: Autophagy is involved in nasopharyngeal carcinoma (NPC) radioresistance. Replication protein A 1 (RPA1) and RPA3, substrates of the RPA complex, are potential therapeutic targets for reversing NPC radioresistance. Nevertheless, the role of RPA in autophagy is not adequately understood. This investigation was performed to reveal the cytotoxic mechanism of a pharmacologic RPA inhibitor (RPAi) in NPC cells and the underlying mechanism by which RPAi-mediated autophagy regulates NPC radiosensitivity. METHODS AND RESULTS: We characterized a potent RPAi (HAMNO) that was substantially correlated with radiosensitivity enhancement and proliferative inhibition of in vivo and in NPC cell lines in vitro. We show that the RPAi induced autophagy at multiple levels by inducing autophagic flux, AMPK/mTOR pathway activation, and autophagy-related gene transcription by decreasing glycolytic function. We hypothesized that RPA inhibition impaired glycolysis and increased NPC dependence on autophagy. We further demonstrated that combining autophagy inhibition with chloroquine (CQ) treatment or genetic inhibition of the autophagy regulator ATG5 and RPAi treatment was more effective than either approach alone in enhancing the antitumor response of NPC to radiation. CONCLUSIONS: Our study suggests that HAMNO is a potent RPAi that enhances radiosensitivity and induces autophagy in NPC cell lines by decreasing glycolytic function and activating autophagy-related genes. We suggest a novel treatment strategy in which pharmacological inhibitors that simultaneously disrupt RPA and autophagic processes improve NPC responsiveness to radiation.

Distinct RPA functions promote eukaryotic DNA replication initiation and elongation.

Replication protein A (RPA) binds single-stranded DNA (ssDNA) and serves critical functions in eukaryotic DNA replication, the DNA damage response, and DNA repair. During DNA replication, RPA is required for extended origin DNA unwinding and DNA synthesis. To determine the requirements for RPA during these processes, we tested ssDNA-binding proteins (SSBs) from different domains of life in reconstituted Saccharomyces cerevisiae origin unwinding and DNA replication reactions. Interestingly, Escherichia coli SSB, but not T4 bacteriophage Gp32, fully substitutes for RPA in promoting origin DNA unwinding. Using RPA mutants, we demonstrated that specific ssDNA-binding properties of RPA are required for origin unwinding but that its protein-interaction domains are dispensable. In contrast, we found that each of these auxiliary RPA domains have distinct functions at the eukaryotic replication fork. The Rfa1 OB-F domain negatively regulates lagging-strand synthesis, while the Rfa2 winged-helix domain stimulates nascent strand initiation. Together, our findings reveal a requirement for specific modes of ssDNA binding in the transition to extensive origin DNA unwinding and identify RPA domains that differentially impact replication fork function.

RBM14 promotes DNA end resection during homologous recombination repair.

DNA double-strand break (DSB) repair by homologous recombination (HR) is crucial for the maintenance of genome stability and integrity. In this study, we aim to identify novel RNA binding proteins (RBPs) involved in HR repair because little is known about RBP function in HR. For this purpose, we carry out pulldown assays using a synthetic ssDNA/dsDNA structure coated with replication protein A (RPA) to mimic resected DNA, a crucial intermediate in HR-mediated DSB repair. Using this approach, we identify RNA-binding motif protein 14 (RBM14) as a potential binding partner. We further show that RBM14 interacts with an essential HR repair factor, CtIP. RBM14 is crucial for CtIP recruitment to DSB sites and for subsequent RPA coating and RAD51 replacement, facilitating efficient HR repair. Moreover, inhibition of RBM14 expression sensitizes cancer cells to X-ray irradiation. Together, our results demonstrate that RBM14 promotes DNA end resection to ensure HR repair and may serve as a potential target for cancer therapy.

UBQLN1 deficiency mediates telomere shortening and IPF through interacting with RPA1.

Premature telomere shortening is a known factor correlated to idiopathic pulmonary fibrosis (IPF) occurrence, which is a chronic, progressive, age-related disease with high mortality. The etiology of IPF is still unknown. Here, we found that UBQLN1 plays a key role in telomere length maintenance and is potentially relevant to IPF. UBQLN1 involves in DNA replication by interacting with RPA1 and shuttling it off from the replication fork. The deficiency of UBQLN1 retains RPA1 at replication fork, hinders replication and thus causes cell cycle arrest and genome instability. Especially at telomere regions of the genome, where more endogenous replication stress exists because of G rich sequences, UBQLN1 depletion leads to rapid telomere shortening in HeLa cells. It revealed that UBQLN1 depletion also shortens telomere length at mouse lung and accelerates mouse lung fibrosis. In addition, the UBQLN1 expression level in IPF patients is downregulated and correlated to poor prognosis. Altogether, these results uncover a new role of UBQLN1 in ensuring DNA replication and maintaining telomere stability, which may shed light on IPF pathogenesis and prevention.

ssDNA accessibility of Rad51 is regulated by orchestrating multiple RPA dynamics.

The eukaryotic single-stranded DNA (ssDNA)-binding protein Replication Protein A (RPA) plays a crucial role in various DNA metabolic pathways, including DNA replication and repair, by dynamically associating with ssDNA. While the binding of a single RPA molecule to ssDNA has been thoroughly studied, the accessibility of ssDNA is largely governed by the bimolecular behavior of RPA, the biophysical nature of which remains unclear. In this study, we develop a three-step low-complexity ssDNA Curtains method, which, when combined with biochemical assays and a Markov chain model in non-equilibrium physics, allow us to decipher the dynamics of multiple RPA binding to long ssDNA. Interestingly, our results suggest that Rad52, the mediator protein, can modulate the ssDNA accessibility of Rad51, which is nucleated on RPA coated ssDNA through dynamic ssDNA exposure between neighboring RPA molecules. We find that this process is controlled by the shifting between the protection mode and action mode of RPA ssDNA binding, where tighter RPA spacing and lower ssDNA accessibility are favored under RPA protection mode, which can be facilitated by the Rfa2 WH domain and inhibited by Rad52 RPA interaction.

Investigating the novel-binding site of RPA2 on Menin and predicting the effect of point mutation of Menin through protein-protein interactions.

Protein-protein interactions (PPIs) play a critical role in all biological processes. Menin is tumor suppressor protein, mutated in multiple endocrine neoplasia type 1 syndrome and has been shown to interact with multiple transcription factors including (RPA2) subunit of replication protein A (RPA). RPA2, heterotrimeric protein required for DNA repair, recombination and replication. However, it's still remains unclear the specific amino acid residues that have been involved in Menin-RPA2 interaction. Thus, accurately predicting the specific amino acid involved in interaction and effects of MEN1 mutations on biological systems is of great interests. The experimental approaches for identifying amino acids in menin-RPA2 interactions are expensive, time-consuming, and challenging. This study leverages computational tools, free energy decomposition and configurational entropy scheme to annotate the menin-RPA2 interaction and effect on menin point mutation, thereby proposing a viable model of menin-RPA2 interaction. The menin-RPA2 interaction pattern was calculated on the basis of different 3D structures of menin and RPA2 complexes, constructed using homology modeling and docking strategy, generating three best-fit models: Model 8 (- 74.89 kJ/mol), Model 28 (- 92.04 kJ/mol) and Model 9 (- 100.4 kJ/mol). The molecular dynamic (MD) was performed for 200 ns and binding free energies and energy decomposition analysis were calculated using Molecular Mechanics Poisson-Boltzmann Surface Area (MM/PBSA) in GROMACS. From binding free energy change, model 8 of Menin-RPA2 exhibited most negative binding energy of - 205.624 kJ/mol, followed by model 28 of Menin-RPA2 with - 177.382 kJ/mol. After S606F point mutation in Menin, increase of BFE (ΔGbind) by - 34.09 kJ/mol in Model 8 of mutant Menin-RPA2 occurs. Interestingly, we found a significant reduction of BFE (ΔGbind) and configurational entropy by - 97.54 kJ/mol and - 2618 kJ/mol in mutant model 28 as compared the o wild type. Collectively, this is the first study to highlight the configurational entropy of protein-protein interactions thereby strengthening the prediction of two significant important interaction sites in menin for the binding of RPA2. These predicted sites could be vulnerable for structural alternation in terms of binding free energy and configurational entropy after missense mutation in menin.

Proper RPA acetylation promotes accurate DNA replication and repair.

The single-stranded DNA (ssDNA) binding protein complex RPA plays a critical role in promoting DNA replication and multiple DNA repair pathways. However, how RPA is regulated to achieve its functions precisely in these processes remains elusive. Here, we found that proper acetylation and deacetylation of RPA are required to regulate RPA function in promoting high-fidelity DNA replication and repair. We show that yeast RPA is acetylated on multiple conserved lysines by the acetyltransferase NuA4 upon DNA damage. Mimicking constitutive RPA acetylation or blocking its acetylation causes spontaneous mutations with the signature of micro-homology-mediated large deletions or insertions. In parallel, improper RPA acetylation/deacetylation impairs DNA double-strand break (DSB) repair by the accurate gene conversion or break-induced replication while increasing the error-prone repair by single-strand annealing or alternative end joining. Mechanistically, we show that proper acetylation and deacetylation of RPA ensure its normal nuclear localization and ssDNA binding ability. Importantly, mutation of the equivalent residues in human RPA1 also impairs RPA binding on ssDNA, leading to attenuated RAD51 loading and homologous recombination repair. Thus, timely RPA acetylation and deacetylation likely represent a conserved mechanism promoting high-fidelity replication and repair while discriminating the error-prone repair mechanisms in eukaryotes.

The RPA-RNF20-SNF2H cascade promotes proper chromosome segregation and homologous recombination repair.

The human tumor suppressor Ring finger protein 20 (RNF20)-mediated histone H2B monoubiquitination (H2Bub) is essential for proper chromosome segregation and DNA repair. However, what is the precise function and mechanism of RNF20-H2Bub in chromosome segregation and how this pathway is activated to preserve genome stability remain unknown. Here, we show that the single-strand DNA-binding factor Replication protein A (RPA) interacts with RNF20 mainly in the S and G2/M phases and recruits RNF20 to mitotic centromeres in a centromeric R-loop-dependent manner. In parallel, RPA recruits RNF20 to chromosomal breaks upon DNA damage. Disruption of the RPA-RNF20 interaction or depletion of RNF20 increases mitotic lagging chromosomes and chromosome bridges and impairs BRCA1 and RAD51 loading and homologous recombination repair, leading to elevated chromosome breaks, genome instability, and sensitivities to DNA-damaging agents. Mechanistically, the RPA-RNF20 pathway promotes local H2Bub, H3K4 dimethylation, and subsequent SNF2H recruitment, ensuring proper Aurora B kinase activation at centromeres and efficient loading of repair proteins at DNA breaks. Thus, the RPA-RNF20-SNF2H cascade plays a broad role in preserving genome stability by coupling H2Bub to chromosome segregation and DNA repair.

Single-molecule visualization of Pif1 helicase translocation on single-stranded DNA.

Pif1 is a broadly conserved helicase that is essential for genome integrity and participates in numerous aspects of DNA metabolism, including telomere length regulation, Okazaki fragment maturation, replication fork progression through difficult-to-replicate sites, replication fork convergence, and break-induced replication. However, details of its translocation properties and the importance of amino acids residues implicated in DNA binding remain unclear. Here, we use total internal reflection fluorescence microscopy with single-molecule DNA curtain assays to directly observe the movement of fluorescently tagged Saccharomyces cerevisiae Pif1 on single-stranded DNA (ssDNA) substrates. We find that Pif1 binds tightly to ssDNA and translocates very rapidly (∼350 nucleotides per second) in the 5'→3' direction over relatively long distances (∼29,500 nucleotides). Surprisingly, we show the ssDNA-binding protein replication protein A inhibits Pif1 activity in both bulk biochemical and single-molecule measurements. However, we demonstrate Pif1 can strip replication protein A from ssDNA, allowing subsequent molecules of Pif1 to translocate unimpeded. We also assess the functional attributes of several Pif1 mutations predicted to impair contact with the ssDNA substrate. Taken together, our findings highlight the functional importance of these amino acid residues in coordinating the movement of Pif1 along ssDNA.

Genomes of the autonomous parvovirus minute virus of mice induce replication stress through RPA exhaustion.

The oncolytic autonomous parvovirus Minute Virus of Mice (MVM) establishes infection in the nuclear environment by usurping host DNA damage signaling proteins in the vicinity of cellular DNA break sites. MVM replication induces a global cellular DNA Damage Response (DDR) that is dependent on signaling by the ATM kinase and inactivates the cellular ATR-kinase pathway. However, the mechanism of how MVM generates cellular DNA breaks remains unknown. Using single molecule DNA Fiber Analysis, we have discovered that MVM infection leads to a shortening of host replication forks as infection progresses, as well as induction of replication stress prior to the initiation of virus replication. Ectopically expressed viral non-structural proteins NS1 and NS2 are sufficient to cause host-cell replication stress, as is the presence of UV-inactivated non-replicative MVM genomes. The host single-stranded DNA binding protein Replication Protein A (RPA) associates with the UV-inactivated MVM genomes, suggesting MVM genomes might serve as a sink for cellular stores of RPA. Overexpressing RPA in host cells prior to UV-MVM infection rescues DNA fiber lengths and increases MVM replication, confirming that MVM genomes deplete RPA stores to cause replication stress. Together, these results indicate that parvovirus genomes induce replication stress through RPA exhaustion, rendering the host genome vulnerable to additional DNA breaks.

Actin nucleators safeguard replication forks by limiting nascent strand degradation.

Accurate genome replication is essential for all life and a key mechanism of disease prevention, underpinned by the ability of cells to respond to replicative stress (RS) and protect replication forks. These responses rely on the formation of Replication Protein A (RPA)-single stranded (ss) DNA complexes, yet this process remains largely uncharacterized. Here, we establish that actin nucleation-promoting factors (NPFs) associate with replication forks, promote efficient DNA replication and facilitate association of RPA with ssDNA at sites of RS. Accordingly, their loss leads to deprotection of ssDNA at perturbed forks, impaired ATR activation, global replication defects and fork collapse. Supplying an excess of RPA restores RPA foci formation and fork protection, suggesting a chaperoning role for actin nucleators (ANs) (i.e. Arp2/3, DIAPH1) and NPFs (i.e, WASp, N-WASp) in regulating RPA availability upon RS. We also discover that ß-actin interacts with RPA directly in vitro, and in vivo a hyper-depolymerizing ß-actin mutant displays a heightened association with RPA and the same dysfunctional replication phenotypes as loss of ANs/NPFs, which contrasts with the phenotype of a hyper-polymerizing ß-actin mutant. Thus, we identify components of actin polymerization pathways that are essential for preventing ectopic nucleolytic degradation of perturbed forks by modulating RPA activity.

Comprehensive mutational analysis of the checkpoint signaling function of Rpa1/Ssb1 in fission yeast.

Replication protein A (RPA) is a heterotrimeric complex and the major single-strand DNA (ssDNA) binding protein in eukaryotes. It plays important roles in DNA replication, repair, recombination, telomere maintenance, and checkpoint signaling. Because RPA is essential for cell survival, understanding its checkpoint signaling function in cells has been challenging. Several RPA mutants have been reported previously in fission yeast. None of them, however, has a defined checkpoint defect. A separation-of-function mutant of RPA, if identified, would provide significant insights into the checkpoint initiation mechanisms. We have explored this possibility and carried out an extensive genetic screen for Rpa1/Ssb1, the large subunit of RPA in fission yeast, looking for mutants with defects in checkpoint signaling. This screen has identified twenty-five primary mutants that are sensitive to genotoxins. Among these mutants, two have been confirmed partially defective in checkpoint signaling primarily at the replication fork, not the DNA damage site. The remaining mutants are likely defective in other functions such as DNA repair or telomere maintenance. Our screened mutants, therefore, provide a valuable tool for future dissection of the multiple functions of RPA in fission yeast.

A DNA damage-induced phosphorylation circuit enhances Mec1ATR Ddc2ATRIP recruitment to Replication Protein A.

The cell cycle checkpoint kinase Mec1ATR and its integral partner Ddc2ATRIP are vital for the DNA damage and replication stress response. Mec1-Ddc2 "senses" single-stranded DNA (ssDNA) by being recruited to the ssDNA binding Replication Protein A (RPA) via Ddc2. In this study, we show that a DNA damage-induced phosphorylation circuit modulates checkpoint recruitment and function. We demonstrate that Ddc2-RPA interactions modulate the association between RPA and ssDNA and that Rfa1-phosphorylation aids in the further recruitment of Mec1-Ddc2. We also uncover an underappreciated role for Ddc2 phosphorylation that enhances its recruitment to RPA-ssDNA that is important for the DNA damage checkpoint in yeast. The crystal structure of a phosphorylated Ddc2 peptide in complex with its RPA interaction domain provides molecular details of how checkpoint recruitment is enhanced, which involves Zn2+. Using electron microscopy and structural modeling approaches, we propose that Mec1-Ddc2 complexes can form higher order assemblies with RPA when Ddc2 is phosphorylated. Together, our results provide insight into Mec1 recruitment and suggest that formation of supramolecular complexes of RPA and Mec1-Ddc2, modulated by phosphorylation, would allow for rapid clustering of damage foci to promote checkpoint signaling.