The human single-strand DNA binding protein RPA rapidly associates with DNA breaks in vivo. I. Pasic^{1, 2}, M.S. Meyn^{1, 2}. 1) Genetics & Genomic Biology, Hosp for Sick Children; 2) Molecular & Medical Genetics, Univ of Toronto, Toronto, ON.
   RPA is a single-strand DNA-binding protein involved in DNA metabolism. In response to DNA double-strand breaks (DSBs), its RPA2 subunit is phosphorylated by the damage response kinase ATM. While phosphorylation alters RPA's interactions with replication and repair factors, it is unclear how it affects interactions between RPA and DSBs. We therefore studied the effect of ATM deficiency on recruitment of RPA2 to photo-induced DSBs in human fibroblasts.
   Endogenous RPA2 is detectable at DSBs by 30s and remains localized to DSBs 150s post-irradiation. RPA2 phosphorylated at T21 (T21~P) is also present at DSBs 30s post-irradiation, but by 90s is diffusely distributed throughout the nuclei of irradiated cells. This suggests that unphosphorylated RPA2 rapidly associates with DSBs, where it is then phosphorylated, thereby promoting RPA dissociation from DSBs. In support of this notion, the association of a T21DS33D phosphomimetic GFP-RPA2 mutant with induced DSBs is impaired and a T21DS23DS33D phosphomimetic GFP-RPA2 mutant associates with DSBs very poorly.
   To further examine the negative role for phosphorylation in control of RPA's association with DSBs, we studied the association of RPA2 with DSBs in ATM-deficient cells. While RPA2 is seen at DSBs in ATM-deficient cells 30s post-irradiation, RPA2-T21~P is not detectable, suggesting DSB-induced phosphorylation of RPA2 at T21 is ATM-dependent, but T21 phosphorylation is unnecessary for RPA2s association with DSBs. Interestingly, in the absence of ATM, GFP-RPA2 associates with DSBs more slowly than in wild type cells. This may reflect a defect in an early processing step needed for RPA2 to associate with DSBs rather than lack of RPA2 phosphorylation. Such a step may involve the MRN complex, which promotes nucleolytic processing of DSBs. In support for this idea, we find that the association of GFP-RPA2 to DSBs in NBS1-deficient cells is severely impaired. Our results suggest that the association of RPA with DSBs is rapid and MRN-dependent; enhanced by ATM-dependent activation of MRN; and decreased by phosphorylation of RPA2, which likely occurs at DSB sites.