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SSRP1 Cooperates with PARP and XRCC1 to Facilitate Single-Strand

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SSRP1 Cooperates with PARP and XRCC1 to Facilitate Single-Strand Published OnlineFirst April 17, 2017; DOI: 10.1158/0008-5472.CAN-16-3128 Cancer Molecular and Cellular Pathobiology Research SSRP1 Cooperates with PARP and XRCC1 to Facilitate Single-Strand DNA Break Repair by Chromatin Priming Ying Gao1,2,3, Changling Li2,3,4, Leizhen Wei2,3, Yaqun Teng1,2,3, Satoshi Nakajima2,3, Xiukai Chen2,3, Jianquan Xu2,5,6, Brittany Leger2, Hongqiang Ma2,5,6, Stephen T. Spagnol7, Yong Wan2,8, Kris Noel Dahl7,9, Yang Liu2,5,6, Arthur S. Levine2,3, and Li Lan2,3 Abstract DNA single-strand breaks (SSB) are the most common form PARP-dependent manner and retained at DNA damage sites of DNA damage, requiring repair processes that to initiate by N-terminal interactions with the DNA repair protein must overcome chromatin barriers. The FACT complex com- XRCC1. Mutational analyses showed how SSRP1 function is prised of the SSRP1 and SPT16 proteins is important for essential for chromatin decondensation and histone H2B maintaining chromatin integrity, with SSRP1 acting as an exchange at sites of DNA strand breaks, which are both critical histone H2A/H2B chaperone in chromatin disassembly during to prime chromatin for efficient SSB repair and cell survival. DNA transcription, replication, and repair. In this study, we By establishing how SSRP1 facilitates SSB repair, our findings show that SSRP1, but not SPT16, is critical for cell survival provide a mechanistic rationale to target SSRP1 as a general after ionizing radiation or methyl methanesulfonate–induced approach to selectively attack cancer cells. Cancer Res; 77(10); single-strand DNA damage. SSRP1 is recruited to SSB in a 2674–85. Ó2017 AACR. Introduction ADP-ribose (PAR), modifying itself as well as other target proteins. PAR may help trigger the relaxation of chromatin around SSBs and DNA single-strand breaks (SSB) arise tens of thousands of times promote the accumulation of X-ray repair cross-complementing per cell per day, resulting from environmental or endogenous protein 1 (XRCC1) and other SSBR proteins at the opened chro- agents such as reactive oxygen species. Unrepaired SSBs in prolif- matin site (3, 4). Targeting PARP activation has been shown as an erating cells will most likely collapse DNA replication forks and effective cancer therapeutic approach; meanwhile, rational com- form DNA double-strand breaks (DSB), leading to genome insta- binations exploiting alternate mechanisms of defective DNA bility (1). DSBs, the most lethal type of DNA damage, are repaired repair, engaging additional functions of PARP, and identifying the by homologous recombination (HR) or nonhomologous end optimal combination partner will present novel opportunities for joining in human cells. In nonproliferating cells, such as post- further treatment options for patients (5). XRCC1, a scaffold mitotic neurons with high transcriptional demand, SSBs lead to protein in SSBR, also physically interacts with PARPs (PARP1 and stalled transcription and trigger cell death (2). Most SSBs can be PARP2), APEI, and other repair factors, thereby organizing SSBR in removed by global SSB repair (SSBR), which includes four major cells in a temporal and spatial manner (6). steps: SSB recognition/detection, DNA end processing, gap filling, TheDNAdamageresponseincellsisreferredtoasthe and ligation (2). PARP1 recognizes SSBs and synthesizes poly- "Access/Prime-Repair-Restore" process for successful repair (7). Access and restore require the disassembly and reassembly 1School of Medicine, Tsinghua University, Beijing, China. 2University of Pitts- of the chromatin structure around damage sites, which involves burgh Cancer Institute, Pittsburgh, Pennsylvania. 3Department of Microbiology chromatin remodeling factors and histone chaperones (8, 9). and Molecular Genetics, University of Pittsburgh School of Medicine, Pittsburgh, The high-mobility group (HMG)-1–like structure-specificrec- Pennsylvania. 4Department of Experimental Medicine, General Hospital of 5 ognition protein-1 (SSRP1) is one of the critical components of Shenyang Military Area Command, Shenyang, Liaoning, China. Department of the Facilitate Chromatin Transcription (FACT) complex togeth- Medicine, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania. 6Department of Bioengineering, University of Pittsburgh Swanson School of er with suppressor of Ty homolog-16 (SPT16; ref. 10), which Engineering, Pittsburgh, Pennsylvania. 7Department of Chemical Engineering, also has been implicated in cancers (11, 12). FACT is a histone Carnegie Mellon University, Pittsburgh, Pennsylvania. 8Department of Cell H2A/H2B chaperone that regulates chromatin dynamics during Biology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania. transcription (13), replication (14, 15), and DNA damage 9 Department of Biomedical Engineering, Carnegie Mellon University, Pittsburgh, repair. FACT is known to mediate the turnover of histone Pennsylvania. H2A/H2A.X, which is regulated by both the phosphorylation Corresponding Author: Li Lan, University of Pittsburgh School of Medicine, 2.6 of H2A.X and the ADP-ribosylation of SPT16 in vitro (16). FACT Hillman Cancer Center, 5117 Centre Avenue, Pittsburgh, PA 15213. Phone: 412- promotes transcription restart by enhancing histone H2A/H2B 623-3228; Fax: 412-623-7766; E-mail: [email protected] exchange at UV damage sites, driven by the larger component doi: 10.1158/0008-5472.CAN-16-3128 SPT16 but not SSRP1 (17). SPT16 also remodels chromatin Ó2017 American Association for Cancer Research. through interaction with RNF20 upon DNA damage to 2674 Cancer Res; 77(10) May 15, 2017 Downloaded from cancerres.aacrjournals.org on September 25, 2021. © 2017 American Association for Cancer Research. Published OnlineFirst April 17, 2017; DOI: 10.1158/0008-5472.CAN-16-3128 SSRP1 Facilitates Chromatin Priming upon Damage promote HR (18). Although SSRP1 is not involved in histone described experiments, we cultured the cells stocked in nitrogen exchange upon UVC-induced damage (17), SSRP1 interacts liquid tank for 3 to 4 weeks. Number of passages are varying from with cisplatin-damaged DNA (19). In addition, SSRP1 deple- 3 to 10. The cell lines were tested by a mycoplasma testing kit tion is associated with increased Rad51 foci, which indicates (AccuSEQ Thermo Fisher Scientific) to exclude the possibility of that SSRP1 is necessary for efficient HR (20). It is not known mycoplasma contamination. All cell lines were cultured in DMEM whether SSRP1 also plays a direct role in repairing the most (Lonza) with 10% FBS (Atlanta Biologicals) at 37 C and 5% CO2. frequent type of DNA damage, SSBs. In this study, we eluci- The siRNAs were transfected into HeLa cells using DharmaFECT1 dated the molecular pathways that explain how SSRP1 is in- (Thermo) in the colony-forming assay. The SSRP1 siRNA (h) (SC- volved in SSB repair and promotion of chromatin priming at 37877) (A: Sense: GCAAGACCUUUGACUACAAtt, Antisense: damage sites so as to facilitate efficient SSBR. We show that UUGUAGUCAAAGGUCUUGCtt; B: Sense: CGUUGACUCU- SSRP1 accumulates at SSBs in a PAR-dependent manner. How GAACAUGAAtt, Antisense: UUCAUGUUCAGAGUCAACGtt; C: SSRP1 remains at damage sites by interacting with XRCC1 is Sense: GGAUCCAAAUCCUCAUCUUtt, Antisense: AAGAUGAG- shown based on an ALFFSRI Rev1 Interacting Region (RIR) GAUUUGGAUCCtt) and SPT16 siRNA (h) (SC-37875) (A: Sense: motif–mediated interaction. Finally, the role of SSRP1 as a GAAGAGCACAUCAGAAAGAtt, Antisense: UCUUUCUGAU- histone chaperone, priming the chromatin around damage sites GUGCUCUUCtt; B: Sense: GUCAUUGGGUAGUGAAGAAtt, for successful SSBR, thus will be a potential cancer treatment Antisense: UUCUUCACUACCCAAUGACtt; C: Sense: GAUGG- target and is discussed. CUUCUGACAUCUUAtt, Antisense: UAAGAUGUCAGAAGC- CAUCtt) were purchased from Santa Cruz Biotechnology. shRNAs Materials and Methods of SSRP1 (human) (SHCLNG-NM_003146) were purchased from Plasmids, transfections, and chemicals Sigma Aldrich, including shSSRP1-1, TRCN0000019271; SSRP1, Histone H2B cDNAs, and the deletions were amplified shSSRP1-2, TRCN0000343894. The shCtrl stably expressed the using XhoI/SalI and NotI, and then subcloned into pEGFP-C1 vector Plko.1 (Sigma mission). (Clonetech). RFP-XRCC1, Flag-XRCC1, and the XRCC1 deletions Microscope and laser microirradiation were cloned previously (21). Cherry-H2B was purchased from The Olympus FV/1000 confocal microscopy system (Olym- Addgene. Plasmids were transfected into cells using Lipofecta- pus) and FV/1000 software were used for image acquisition and mine 2000 (Invitrogen) according to the manufacturer's instruc- analysis and described in a previous study (22). Damage was tions. Olaparib (10 nmol/L, Catalog Nos. A4154, APExBIO), induced with a 405 nm laser. The laser passed through a ABT88 (10 nmol/L, APExBio), and PJ34 (20 nmol/L, Sigma) were PLAON 60X oil lens. The output power was 5 mW/scan. Cells used in imaging and immunoprecipitation (IP) as well as for transfected with GFP-tagged proteins were incubated at 37C survival assays. MMS (129925-5G, Sigma) was used with the on a thermos plate in normal media during observation. For indicated dose in survival assays. Bleocin (CALBIOCHEM, 5 mg/ the evaluation of accumulation and kinetics, the mean intensity mL, 1:1,000) was used to induce damage in HeLa cells. of each accumulated point or line was obtained after subtrac- fi Site-directed mutagenesis tion and quanti ed by ImageJ. The N-terminal of XRCC1 was subcloned from pEGFP-C1- XPA-UVDE system induced DNA SSBs after local UVC XRCC1 into the plasmid of pBlueScript KS (þ) via Sal I and
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