NBS1 mediates ATR-dependent RPA hyperphosphorylation following replication-fork stall and collapse

Abstract

Post-translational phosphorylation of proteins provides a mechanism for cells to switch on or off many diverse processes, including responses to replication stress. Replication-stress-induced phosphorylation enables the rapid activation of numerous proteins involved in DNA replication, DNA repair and cell cycle checkpoints, including replication protein A (RPA). Here, we report that hydroxyurea (HU)-induced RPA phosphorylation requires both NBS1 (NBN) and NBS1 phosphorylation. Transfection of both phosphospecific and nonphosphospecific anti-NBS1 antibodies blocked hyperphosphorylation of RPA in HeLa cells. Nijmegen breakage syndrome (NBS) cells stably transfected with an empty vector or with S343A-NBS1 or S278A/S343A phospho-mutants were unable to hyperphosphorylate RPA in DNA-damage-associated foci following HU treatment. The stable transfection of fully functional NBS1 in NBS cells restored RPA hyperphosphorylation. Retention of ATR on chromatin in both NBS cells and in NBS cells expressing S278A/S343A NBS1 mutants decreased after DNA damage, suggesting that ATR is the kinase responsible for RPA phosphorylation. The importance of RPA hyperphosphorylation is demonstrated by the ability of cells expressing a phospho-mutant form of RPA32 (RPA2) to suppress and delay HU-induced apoptosis. Our findings suggest that RPA hyperphosphorylation requires NBS1 and is important for the cellular response to DNA damage.

ATM Protein-dependent Phosphorylation of Rad50 Protein Regulates DNA Repair and Cell Cycle Control

“…These models suggest that NBN acts as a multimodal adaptor linking the MRN complex to ATM and several other proteins involved in the response to DNA DSB (78). Its function is attenuated by phosphorylation in controlling the S-phase checkpoint (44,63,64) in the down-regulation of DNA replication after UV exposure (79) and in mediating ATR-dependent replication protein A1 hyperphosphorylation during replication fork stalling (80). Although not demonstrated, it seems likely that NBN Ser(P)-343 leads to conformational change that alters the function or interaction of the sub- units of MRN at the site of the DNA DSB.…”

Coronavirus Infection Induces DNA Replication Stress Partly through Interaction of Its Nonstructural Protein 13 with the p125 Subunit of DNA Polymerase δ

“…RPA is a single-strand DNA-binding protein and may play critical roles in DNA synthesis, damage repair, and recombination (55,56). RPA2, a component of the heterotrimeric RPA complex, formed nuclear foci in response to various DNA damage signals including UV irradiation and hydroxyurea treatment (57,58). Formation of nuclear foci by RPA2 would be an additional indicator of DNA damage response in IBV-infected cells.…”

Human RPA phosphorylation by ATR stimulates DNA synthesis and prevents ssDNA accumulation during DNA-replication stress

“…Two of the sites (T21 and S33) are consensus for phosphatidylinositol 3-kinase-like kinase (PIKK)-family members (ATM, ATR and DNA-PK), whereas others are targets of the cyclinCdk complex (S23 and S29) or DNA-PK (S4, S8, S11, S12 and S13; Fig. 1A) (Anantha et al, 2007;Block et al, 2004;Dutta and Stillman, 1992;Fang and Newport, 1993;Liu et al, 2006;Manthey et al, 2007;Niu et al, 1997;Oakley et al, 2003;Olson et al, 2006;Pan et al, 1994;Zernik-Kobak et al, 1997). Treatment of cells with the DNA-damaging agent camptothecin (CPT) causes initial RPA phosphorylation on S33 by ATR; this phosphorylation subsequently stimulates phosphorylation by cyclin-Cdk and DNA-PK to yield hyperphosphorylated RPA (Anantha et al, 2007).…”