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The heterotrimeric Replication protein A (RPA) is the ubiquitous eukaryotic single-stranded DNA (ssDNA) binding protein and participates in nearly all aspects of DNA metabolism, especially DNA damage response. The N-terminal OB domain of the RPA70 subunit (RPA70N) is a major protein-protein interaction element for RPA and binds to more than 20 partner proteins. Previous crystallography studies of RPA70N with p53, DNA2 and PrimPol fragments revealed that RPA70N binds to amphipathic peptides that mimic ssDNA. NMR chemical-shift studies also provided valuable information on the interaction of RPA70N residues with target sequences. However, it is still unclear how RPA70N recognizes and distinguishes such a diverse group of target proteins. Here, we present high-resolution crystal structures of RPA70N in complex with peptides from eight DNA damage response proteins. The structures show that, in addition to the ssDNA mimicry mode of interaction, RPA70N employs multiple ways to bind its partners. Our results advance the mechanistic understanding of RPA70N-mediated recruitment of DNA damage response proteins., Competing Interests: YW, WF, NZ, CZ No competing interests declared, (© 2023, Wu, Fu, Zang et al.)

Cells have evolved the DNA damage response (DDR) pathways in response to DNA replication stress or DNA damage. In the ATR-Chk1 DDR pathway, it has been proposed that ATR is recruited to RPA-coated single-stranded DNA (ssDNA) by direct ATRIP-RPA interaction. However, it remains elusive how ATRIP is recruited to ssDNA in an RPA-independent manner. Here, we provide evidence that APE1 directly associates ssDNA to recruit ATRIP onto ssDNA in an RPA-independent fashion. The N-terminal motif within APE1 is required and sufficient for the APE1-ATRIP interaction in vitro and the distinct APE1-ATRIP interaction is required for ATRIP recruitment to ssDNA and the ATR-Chk1 DDR pathway activation in Xenopus egg extracts. In addition, APE1 directly associates with RPA70 and RPA32 via two distinct motifs. Taken together, our evidence suggests that APE1 recruits ATRIP onto ssDNA in an RPA-dependent and -independent manner in the ATR DDR pathway., Competing Interests: YL, JL, HZ, AM, KM, SY No competing interests declared, (© 2023, Lin et al.)

Replication protein A (RPA) is a major regulator of eukaryotic DNA metabolism involved in multiple essential cellular processes. Maintaining appropriate RPA dynamics is crucial for cells to prevent RPA exhaustion, which can lead to replication fork breakage and replication catastrophe. However, how cells regulate RPA availability during unperturbed replication and in response to stress has not been well elucidated. Here, we show that HNRNPA2B1 SUMO functions as an endogenous inhibitor of RPA during normal replication. HNRNPA2B1 SUMO associates with RPA through recognizing the SUMO-interacting motif (SIM) of RPA to inhibit RPA accumulation at replication forks and impede local ATR activation. Declining HNRNPA2 SUMO induced by DNA damage will release nuclear soluble RPA to localize to chromatin and enable ATR activation. Furthermore, we characterize that HNRNPA2B1 hinders homologous recombination (HR) repair via limiting RPA availability, thus conferring sensitivity to PARP inhibitors. These findings establish HNRNPA2B1 as a critical player in RPA-dependent surveillance networks., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Besides TopBP1, ETAA1 has been identified more recently as an activator of the ATR-ATRIP complex in human cells. We have examined the role of ETAA1 in the Xenopus egg-extract system, which has been instrumental in the study of ATR-ATRIP. Depletion of ETAA1 from egg extracts did not noticeably reduce the activation of ATR-ATRIP in response to replication stress, as monitored by the ATR-dependent phosphorylation of Chk1 and RPA. Moreover, lack of ETAA1 did not appear to affect DNA replication during an unperturbed S-phase. Significantly, we find that TopBP1 is considerably more abundant than ETAA1 in egg extracts. We proceeded to show that ETAA1 could support the activation of ATR-ATRIP in response to replication stress if we increased its concentration in egg extracts by adding extra full-length recombinant ETAA1. Thus, TopBP1 appears to be the predominant activator of ATR-ATRIP in response to replication stress in this system. We have also explored the biochemical mechanism by which ETAA1 activates ATR-ATRIP. We have developed an in vitro system in which full-length recombinant ETAA1 supports activation of ATR-ATRIP in the presence of defined components. We find that binding of ETAA1 to RPA associated with single-stranded DNA (ssDNA) greatly stimulates its ability to activate ATR-ATRIP. Thus, RPA-coated ssDNA serves as a direct positive effector in the ETAA1-mediated activation of ATR-ATRIP.

Mec1-Ddc2 (ATR-ATRIP) is a key DNA-damage-sensing kinase that is recruited through the single-stranded (ss) DNA-binding replication protein A (RPA) to initiate the DNA damage checkpoint response. Activation of ATR-ATRIP in the absence of DNA damage is lethal. Therefore, it is important that damage-specific recruitment precedes kinase activation, which is achieved at least in part by Mec1-Ddc2 homodimerization. Here, we report a structural, biochemical, and functional characterization of the yeast Mec1-Ddc2-RPA assembly. High-resolution co-crystal structures of Ddc2-Rfa1 and Ddc2-Rfa1-t11 (K45E mutant) N termini and of the Ddc2 coiled-coil domain (CCD) provide insight into Mec1-Ddc2 homodimerization and damage-site targeting. Based on our structural and functional findings, we present a Mec1-Ddc2-RPA-ssDNA composite structural model. By way of validation, we show that RPA-dependent recruitment of Mec1-Ddc2 is crucial for maintaining its homodimeric state at ssDNA and that Ddc2's recruitment domain and CCD are important for Mec1-dependent survival of UV-light-induced DNA damage., (Copyright © 2017 Elsevier Inc. All rights reserved.)

The ataxia telangiectasia-mutated and Rad3-related (ATR) kinase checkpoint pathway maintains genome integrity; however, the role of the sirtuin 2 (SIRT2) acetylome in regulating this pathway is not clear. We found that deacetylation of ATR-interacting protein (ATRIP), a regulatory partner of ATR, by SIRT2 potentiates the ATR checkpoint. SIRT2 interacts with and deacetylates ATRIP at lysine 32 (K32) in response to replication stress. SIRT2 deacetylation of ATRIP at K32 drives ATR autophosphorylation and signaling and facilitates DNA replication fork progression and recovery of stalled replication forks. K32 deacetylation by SIRT2 further promotes ATRIP accumulation to DNA damage sites and binding to replication protein A-coated single-stranded DNA (RPA-ssDNA). Collectively, these results support a model in which ATRIP deacetylation by SIRT2 promotes ATR-ATRIP binding to RPA-ssDNA to drive ATR activation and thus facilitate recovery from replication stress, outlining a mechanism by which the ATR checkpoint is regulated by SIRT2 through deacetylation., (Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.)

Abstract: Replication protein A (RPA) interacts with multiple checkpoint proteins and promotes signaling through the ATR kinase, a key regulator of checkpoint pathways in the mammalian response to DNA damage. In cancer cells, increased DNA repair activity contributes to resistance to chemotherapy. Therefore, small molecules that block binding of checkpoint proteins to RPA may inhibit the DNA damage response and, thus, sensitize cancer cells to DNA-damaging agents. Here we report on the development of a homogeneous, high-throughput fluorescence polarization assay for identifying compounds that block the critical protein–protein interaction site in the basic cleft of the 70N domain of RPA (RPA70N). A fluorescein isothiocyanate (FITC)-labeled peptide derived from the ATR cofactor, ATRIP, was used as a probe in the binding assay. The ability of the assay to accurately detect relevant ligands was confirmed using peptides derived from ATRIP, RAD9, MRE11, and p53. The assay was validated for use in high-throughput screening using the Spectrum collection of 2000 compounds. The FPA assay was performed with a Z′ factor of ⩾0.76 in a 384-well format and identified several compounds capable of inhibiting the RPA70N binding interface. [Copyright &y& Elsevier]