XPA plays a role to recruit endonuclease for removing UVC damage in the nucleotide excision repair (NER) pathway. (16). FACT promotes transcription restart by enhancing histone H2A/H2B exchange at UV damage sites, driven by the larger component SPT16 but not SSRP1 (17). SPT16 also remodels chromatin through conversation with RNF20 upon DNA damage to promote HR (18). Although SSRP1 is not involved in histone exchange upon UVC-induced damage (17), SSRP1 interacts with cisplatin-damaged DNA (19). In addition, SSRP1 depletion is usually associated with increased Rad51 foci, which indicates that SSRP1 is necessary for efficient HR (20). It is not known whether SSRP1 also plays a direct role in repairing the most frequent type of DNA damage, SSBs. In this study, we elucidated the molecular pathways of if and how SSRP1 is usually involved in single strand break repair and promotion of chromatin priming at damage sites so as to facilitate efficient SSBR. We show that SSRP1 accumulates at SSBs in a PAR-dependent manner. How SSRP1 remains at damage sites by interacting with XRCC1 is usually shown based on a ALFFSRI RIR motif mediated conversation. Finally, the role of SSRP1 as a histone chaperone, priming the chromatin around damage sites for successful SSBR thus will be a potential cancer treatment target is usually discussed. Experimental procedures Plasmids, transfections, and chemicals SSRP1, Histone H2B cDNAs, and the deletions were amplified using XhoI/SalI and NotI, then subcloned into pEGFP-C1 (Clonetech). RFP-XRCC1, Flag-XRCC1, and the XRCC1 deletions were cloned previously (21). Cherry-H2B was purchased from Addgene. Plasmids were transfected into cells using Lipofectamine 2000 (Invitrogen) according to the manufacturers instructions. Olaparib (10 nM, Catalog No. A4154, APExBIO), ABT88 (10 nM, APExBio), and PJ34 (20 nM, Sigma) were used in imaging and immunoprecipitation (IP) as well as for survival assays. MMS (129925-5G, Sigma) was used with the indicated dose in survival assays. Bleocin (CALBIOCHEM, 5 g/l 1:1000) was used to induce damage in HeLa cells. Site-directed Mutagenesis The N-terminal of was subcloned from pEGFP-C1-XRCC1 into the plasmid of pBlueScript KS (+) via Sal I and Kpn I digestion. Site-directed mutagenesis was performed by polymerase chain reactions (PCR) with mutated pairs of primers and KOD warm start DNA polymerase (71086-3, Novagen, MA, USA), using the subcloned pBS-SK-XRCC1 as the template. After digestion with Dpn I (R0176S, NEB, USA), the mutated plasmids were transformed into TOP10 qualified cells and screened on plates with ampicillin. Subsequently, the isolated plasmid DNA was sent to the Genomic Core Facilities of the University of Pittsburgh for sequencing to further confirm the mutations. Finally, all three mutated genes were transferred back onto the vector of pEGFP-C1 via Sal and KpnI. The mutation primers used are as follows, pBS-XRCC1-F67A-For: 5-GGAATGATGGCTCAGCTGCCGTGGAGGTGCTGGCGGG-3; pBS-XRCC1-F67A-Rev: 5-CCCGCCAGCACCTCCACGGCAGCTGAGCCATCATTCC-3 pBS-XRCC1-FF191/192AA-For:5-CAACTCTCTGAGGCCGGGGGCTCTCgcCgcCAGCCGGATCAACAAGACATCCCCAG-3; and pBS-XRCC1-FF191/192AA-Rev: 5-CTGGGGATGTCTTGTTGATCCGGCTGgcGgcGAGAGCCCCCGGCCTCAGAGAGTTG-3 Cell lines and siRNA/shRNAs U2OS, HeLa, and FLP-in-293 cells were purchased from ATCC in 2012. XPA-C2 and XPA-UVDE Cells was originally derived in Mibefradil Dr. Akira Yasuis lab in 2010 2010, in which a foreign UV damage endonuclease (UVDE) or a control vector was stably introduced into human xeroderma pigmentosum group A (XPA)-deficient cells to make XPA-UVDE or XPA-C2 cells. In the described experiments, we cultured CTG3a the cells stocked in Nitrogen liquid tank for 3C4 weeks. Number of passages are varying from 3C10. The cells lines were tested by mycoplasma testing kit (AccuSEQ Thermo Fisher Scientific) to exclude the possibility of mycoplasma contamination. All cell lines were cultured in Dulbeccos altered es Mibefradil medium (DMEM, Lonza) with 10% fetal bovine serum (Atlanta Biologicals) at 37C and 5% CO2. The siRNAs were transfected into HeLa cells using DharmaFECT1 Mibefradil (Thermo) in the colony-forming assay. The SSRP1 siRNA (h) (SC-37877) (A: Sense: GCAAGACCUUUGACUACAAtt, Antisense: UUGUAGUCAAAGGUCUUGCtt; B: Sense: CGUUGACUCUGAACAUGAAtt, Antisense: UUCAUGUUCAGAGUCAACGtt; C: Sense: GGAUCCAAAUCCUCAUCUUtt, Antisense: AAGAUGAGGAUUUGGAUCCtt) and SPT16 siRNA.