CtIP Links DNA Double-Strand Break Sensing to Resection

Abstract

Summary In response to DNA double-strand breaks (DSBs), cells sense the DNA lesions and then activate the protein kinase ATM. Subsequent DSB resection produces RPA-coated ssDNA that is essential for activation of the DNA damage checkpoint and DNA repair by homologous recombination (HR). However, the biochemical mechanism underlying the transition from DSB sensing to resection remains unclear. Using Xenopus egg extracts and human cells we show that the tumor suppressor protein CtIP plays a critical role in this transition. We find that CtIP translocates to DSBs, which is dependent on the DSB sensor complex Mre11-Rad50-NBS1, the kinase activity of ATM and a direct DNA-binding motif in CtIP, and then promotes DSB resection. Thus, CtIP facilitates the transition from DSB sensing to processing: It does so by binding to the DNA at DSBs after DSB sensing and ATM activation, and then promoting DNA resection leading to checkpoint activation and HR.

DNA-dependent Protein Kinase Regulates DNA End Resection in Concert with Mre11-Rad50-Nbs1 (MRN) and Ataxia Telangiectasia-mutated (ATM)

“…Other reports have shown that ATM actively regulates HR (16,57), and we also observe here that inhibition of ATM leads to a lower rate of resection (Fig. 1B).…”

“…1F) suggested there might be an important role for ATM in regulating DNA-PKcs inhibition of resection. At least two of the phosphorylation sites in the ABCDE cluster, Thr-2609 and Thr-2647, have been identified as ATM target sites in human cells (26), and ATM activity has been shown to be required for resection of DBSs in Xenopus extracts (16) and for a subset of HR in human cells (52). Here, we found that ATM could further alleviate DNA-PKcs inhibition of resection in the presence of MRN (Fig.…”

The distinctive cellular responses to DNA strand breaks caused by a DNA topoisomerase I poison in conjunction with DNA replication and RNA transcription

“…Moreover, observation of Ku nuclear foci with a high-resolution microscope revealed that although Ku barely forms foci in response to CPT, CPT-induced Ku foci can be observed with an ATM inhibitor (Britton et al, 2013). Since ATM inhibition is believed to down-regulate end resection (You et al, 2009), the Ku foci data further support the above model in which Ku is immediately removed from oneended DSBs via ATM-dependent end resection that excludes NHEJ and starts HR.…”

“…ATM and DNA-PKcs are primarily activated in response to DSBs, whereas ATR functions as a major sensing factor in response to DNA replication stress (Cimprich and Cortez, 2008;Lovejoy and Cortez, 2009). ATM is immediately activated by DSB formation and contributes to the HR pathway by promoting chromatin remodeling and the DNA end resection required for subsequent strand exchange (Goodarzi et al, 2008;You et al, 2009), whereas DNA-PKcs senses DSBs as a complex with Ku proteins and promotes synapsis of two DNA ends and the subsequent end-joining reaction (DeFazio et al, 2002;Spagnolo et al, 2006).…”

Protein Phosphorylation in Human Health

“…Three exonucleases (Mre11, Exo1 and CtIP) and one helicase (BLM) have been involved in resection [32][33][34]. Importantly, ATM dependent phosphorylation seems to stimulate CtIPmediated resection, linking DNA damage signaling with the processing of DNA ends [35]. In addition to ATM, ssDNA is quickly coated by RPA (replication protein A) and triggers a parallel pathway resulting in the activation of the kinase ATR [18,[36][37][38]], that will be described below.…”