title: Damage response of XRCC1 at sites of DNA single strand breaks is regulated by phosphorylation and ubiquitylation after degradation of poly(ADP-ribose).
abstract: Single-strand breaks (SSBs) are the most common type of oxidative DNA damage and they are related to aging and many genetic diseases. The scaffold protein for repair of SSBs, XRCC1, accumulates at sites of poly(ADP-ribose) (pAR) synthesized by PARP, but it is retained at sites of SSBs after pAR degradation. How XRCC1 responds to SSBs after pAR degradation and how this affects repair progression are not well understood. We found that XRCC1 dissociates from pAR and is translocated to sites of SSBs dependent on its BRCTII domain and the function of PARG. In addition, phosphorylation of XRCC1 is also required for the proper dissociation kinetics of XRCC1 because (1) phosphorylation sites mutated in XRCC1 (X1 pm) cause retention of XRCC1 at sites of SSB for a longer time compared to wild type XRCC1; and (2) phosphorylation of XRCC1 is required for efficient polyubiquitylation of XRCC1. Interestingly, a mutant of XRCC1, LL360/361DD, which abolishes pAR binding, shows significant upregulation of ubiquitylation, indicating that pARylation of XRCC1 prevents the poly-ubiquitylation. We also found that the dynamics of the repair proteins DNA polymerase beta, PNK, APTX, PCNA and ligase I are regulated by domains of XRCC1. In summary, the dynamic damage response of XRCC1 is regulated in a manner that depends on modifications of polyADP-ribosylation, phosphorylation and ubiquitylation in live cells.
authors.affiliation: Department of Microbiology and Molecular Genetics, University of Pittsburgh School of Medicine, University of Pittsburgh, Pittsburgh, Pennsylvania, 15261, USA.
authors.forename: Leizhen
authors.initials: L
authors.lastname: Wei
authors.forename: Satoshi
authors.initials: S
authors.lastname: Nakajima
authors.forename: Ching-Lung
authors.initials: CL
authors.lastname: Hsieh
authors.forename: Shinichiro
authors.initials: S
authors.lastname: Kanno
authors.forename: Mitsuko
authors.initials: M
authors.lastname: Masutani
authors.forename: Arthur S
authors.initials: AS
authors.lastname: Levine
authors.forename: Akira
authors.initials: A
authors.lastname: Yasui
authors.forename: Li
authors.initials: L
authors.lastname: Lan
mesh_terms: D053904:DNA Breaks, Single-Stranded; D000072481:DNA Ligase ATP; D011088:DNA Ligases / Q000378:metabolism; D004260:DNA Repair; D004268:DNA-Binding Proteins / Q000378:metabolism*; D057809:HEK293 Cells; D006367:HeLa Cells; D006801:Humans; D010766:Phosphorylation; D011065:Poly(ADP-ribose) Polymerases / Q000378:metabolism*; D000075223:Poly-ADP-Ribose Binding Proteins; D017434:Protein Structure, Tertiary; D014162:Transfection; D054875:Ubiquitination; D000076105:X-ray Repair Cross Complementing Protein 1; D029867:Xenopus Proteins
chemical_list: D004268:DNA-Binding Proteins; D000075223:Poly-ADP-Ribose Binding Proteins; D000076105:X-ray Repair Cross Complementing Protein 1; C000619371:XRCC1 protein, human; D029867:Xenopus Proteins; D011065:Poly(ADP-ribose) Polymerases; D011088:DNA Ligases; D000072481:DNA Ligase ATP; C000618816:DNA ligase III alpha protein, Xenopus
keywords: Damage response; Phosphorylation; PolyADP-ribosylation; Single strand breaks; Ubiquitylation; XRCC1