Abstract
Poly (ADP-ribose) is synthesized at DNA single-strand breaks and can promote the recruitment of the scaffold protein, XRCC1. However, the mechanism and importance of this process has been challenged. To address this issue, we have characterized the mechanism of poly (ADP-ribose) binding by XRCC1 and examined its importance for XRCC1 function. We show that the phosphate-binding pocket in the central BRCT1 domain of XRCC1 is required for selective binding to poly (ADP-ribose) at low levels of ADP-ribosylation, and promotes interaction with cellular PARP1. We also show that the phosphate-binding pocket is required for EGFP-XRCC1 accumulation at DNA damage induced by UVA laser, H2O2, and at sites of sub-nuclear PCNA foci, suggesting that poly (ADP-ribose) promotes XRCC1 recruitment both at single-strand breaks globally across the genome and at sites of DNA replication stress. Finally, we show that the phosphate-binding pocket is required following DNA damage for XRCC1-dependent acceleration of DNA single-strand break repair, DNA base excision repair, and cell survival. These data support the hypothesis that poly (ADP-ribose) synthesis promotes XRCC1 recruitment at DNA damage sites and is important for XRCC1 function.
Highlights
(ADP-ribose) is synthesized at DNA singlestrand breaks and can promote the recruitment of the scaffold protein, XRCC1
Several recent reports have challenged the importance of PAR binding for XRCC1 function, instead ascribing XRCC1 recruitment to DNA binding protein partners such as DNA polymerase  (Pol), polynucleotide kinase/phosphatase (PNKP), and DNA ligase III␣ (Lig3␣) [18,19,20,21,22]
The most evolutionary conserved and functionally important region of XRCC1 is the central BRCT1 domain that mediates binding to PAR [28]
Summary
(ADP-ribose) is synthesized at DNA singlestrand breaks and can promote the recruitment of the scaffold protein, XRCC1. We show that the phosphate-binding pocket is required following DNA damage for XRCC1-dependent acceleration of DNA single-strand break repair, DNA base excision repair, and cell survival These data support the hypothesis that poly (ADP-ribose) synthesis promotes XRCC1 recruitment at DNA damage sites and is important for XRCC1 function. Depletion of PARG, the enzyme responsible for PAR degradation following SSBR, increases both steady state cellular levels of PAR and the accumulation and/or persistence of XRCC1 in sub-nuclear foci before and after DNA damage [17] Despite these observations, several recent reports have challenged the importance of PAR binding for XRCC1 function, instead ascribing XRCC1 recruitment to DNA binding protein partners such as DNA polymerase  (Pol), polynucleotide kinase/phosphatase (PNKP), and DNA ligase III␣ (Lig3␣) [18,19,20,21,22].
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