Abstract
SummarySingle-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.
Highlights
Single-strand breaks (SSBs), one of the most common DNA lesions in human cells, cause transcription and replication blocks, leading to genomic instability and cell death (Cooke et al, 2003)
The BRCT II domain is necessary for retention of XRCC1 at sites of SSBs but not at activated poly(ADP-ribose) sites, whereas phosphorylation of XRCC1 is required for its dissociation from SSBs XRCC1 serves as a scaffold protein in the repair of SSBs and forms foci after methylmethane sulfonate (MMS) treatment or H2O2 treatment (El-Khamisy et al, 2003; Lan et al, 2004; Okano et al, 2003)
We and other groups have shown that accumulation of XRCC1 at SSBs is dependent on Poly(ADP-ribose) polymerase (PARP) activation, and the BRCT I domain of XRCC1 is necessary for its accumulation in cells (Lan et al, 2004; Okano et al, 2003)
Summary
Single-strand breaks (SSBs), one of the most common DNA lesions in human cells, cause transcription and replication blocks, leading to genomic instability and cell death (Cooke et al, 2003). Poly(ADP-ribose) polymerase (PARP) is a nick sensor and binds to SSBs, short gaps in duplex DNA, DSBs and other abnormal DNA structures and initiates the efficient repair of SSBs (Caldecott, 2003; Lan et al, 2004; Oei et al, 2005; Okano et al, 2003). After activation of PARP at SSBs, XRCC1 accumulates at poly(ADP-ribose) (pAR) sites (El-Khamisy et al, 2003; Lan et al, 2004; Okano et al, 2003). After degradation of pAR, XRCC1 appears to act as a molecular scaffold or matchmaker, recruiting and regulating the enzymatic components of the repair process at various stages of SSBR, but the key to retention of XRCC1 at sites of DNA damage is not known
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