Abstract
The primary eukaryotic single-stranded DNA-binding protein, Replication protein A (RPA), binds to single-stranded DNA at the sites of DNA damage and recruits the apical checkpoint kinase, ATR via its partner protein, ATRIP. It has been demonstrated that absence of RPA incapacitates the ATR-mediated checkpoint response. We report that in the absence of RPA, human single-stranded DNA-binding protein 1 (hSSB1) and its partner protein INTS3 form sub-nuclear foci, associate with the ATR-ATRIP complex and recruit it to the sites of genomic stress. The ATRIP foci formed after RPA depletion are abrogated in the absence of INTS3, establishing that hSSB-INTS3 complex recruits the ATR-ATRIP checkpoint complex to the sites of genomic stress. Depletion of homologs hSSB1/2 and INTS3 in RPA-deficient cells attenuates Chk1 phosphorylation, indicating that the cells are debilitated in responding to stress. We have identified that TopBP1 and the Rad9-Rad1-Hus1 complex are essential for the alternate mode of ATR activation. In summation, we report that the single-stranded DNA-binding protein complex, hSSB1/2-INTS3 can recruit the checkpoint complex to initiate ATR signaling.
Highlights
Exposure to genomic insults causes the activation of apical checkpoint kinases, Ataxia telangiectasia mutated (ATM) and Ataxia telangiectasia and Rad3-related protein (ATR)
RPA70 depletion leads to S-phase accumulation and in order to verify whether RPA70 deficiency-induced Chk1 phosphorylation occurs during the S-phase, we utilized mevastatin to block the cells in G1-phase and carried out RPA70 small interfering RNA (siRNA) transfection (Figure 1C) [31]
The N-terminus OB fold of RPA70 binds to ATR-interacting protein (ATRIP), the centrally located OB folds bind to single-stranded DNA (ssDNA) while the Cterminus OB fold interacts with RPA32, which assembles the complex by binding to RPA14 [37,38,39]
Summary
Exposure to genomic insults causes the activation of apical checkpoint kinases, Ataxia telangiectasia mutated (ATM) and Ataxia telangiectasia and Rad3-related protein (ATR). It was demonstrated that Chk is phosphorylated by ATR in vitro and UV-induced phosphorylation of Chk is reduced in cells expressing kinase-inactive ATR. In response to genotoxic agents, Chk was phosphorylated on Serine 317 and 345 in an ATR-dependent manner and mutations at these residues resulted in poor Chk activation [4]. These observations establish that exposure to genotoxic agents results in ATR-mediated phosphorylation of Chk
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
