Work is underway to test this possibility. == A DNA-binding motif in CtIP mediates FGFR4-IN-1 its damage recruitment == By monitoring the recruitment of GFP-tagged CtIP mutant proteins to laser-induced DSBs, we have identified a 49 amino acid DR motif in human CtIP that can recruit fused GFP to DNA damage sites (Fig. 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. == Introduction == The ability of cells to detect and properly repair DNA DSBs is essential for maintaining genome stability and preventing cancer (Kastan and Bartek, 2004). DSBs are generated by ionizing radiation, perturbations of DNA replication and many cancer therapeutic drugs. Eukaryotic cells have evolved a highly conserved checkpoint mechanism to sense and signal the existence of DSBs (Harper and Elledge, 2007). Central to the DSB checkpoint response are the ATM/ATR protein kinases, which receive signals from DNA damage sensors and then undergo activation (Cimprich and Cortez, 2008;Lee and Paull, 2007). Following activation, ATM and ATR phosphorylate a variety of downstream substrates to initiate both local responses at damage sites, FGFR4-IN-1 such as chromatin modification and DNA repair, and global responses, such as cell cycle arrest, transcriptional changes and apoptosis (Kurz and Lees-Miller, 2004;Matsuoka et al., 2007). Although ATM and ATR are related kinases and share many substrates, their activation requires different DNA structures at DSBs. While ATM activation occurs on the DNA/chromatin flanking DNA ends and does not require DNA end resection (You et al., 2007), ATR is activated FGFR4-IN-1 on RPA-coated ssDNA that is generated by 5 > 3 DSB resection (Zou and Elledge, 2003). In addition to activation of ATR, the ssDNA structure generated by DSB resection is also required for formation of the Rad51-ssDNA filament, a key intermediate required for homology probing and D-loop formation during HR repair (Sung and Klein, 2006). Despite its importance, however, the biochemical mechanism and regulation of DSB resection remain poorly understood. InSaccharomyces cerevisiae, genetic studies suggest a 2-step model for Rabbit Polyclonal to GPR108 DSB resection. In this model, the Sae2 protein and the Mre11-Rad50-Xrs2 (MRX) complex (the counterpart of MRN in metazoans) carry out the initial stage of resection to remove a few hundred nucleotides of the 5 strand DNA from a DSB end. Subsequently, the Exo1 exonuclease and the Sgs1/DNA2 helicase/exonuclease act redundantly to further resect 5 strand DNA to generate long 3 ssDNA tails (Budd and Campbell, 2009;Mimitou and Symington, 2008;Zhu et al., 2008). Whether a similar DSB resection model applies in metazoans remains to be determined. Recent studies suggest that the transcriptional regulator/tumor suppressor CtIP in mammals is the functional counterpart of Sae2, despite the very limited sequence similarity between these proteins. Human CtIP is recruited to DNA damage sites and is required for formation of RPA foci at DSBs (indicative of ssDNA generation), and subsequent Chk1 phosphorylation by ATR and HR in response to DSB-inducing agents (Chen et al., 2008;Greenberg et al., 2006;Huertas and Jackson, 2009;Sartori et al., 2007;Yu and Chen, 2004;Yuan and Chen, 2009). Studies on the putative CtIP homologs inS. pombe(Ctp1),C. elegans(COM-1),A. thaliana(AtGR1) and chicken support a role for these proteins in DSB resection (Akamatsu et al., 2008;Limbo et al., 2007;Penkner et al., 2007;Uanschou et al., 2007;Yun and Hiom, 2009). Like Sae2 and Ctp1, CtIP apparently also functions in cooperation with the MRN complex in DSB resection (Chen et al., 2008;Lloyd et al., 2009;Sartori et al., 2007;Williams et al., 2009). In addition to its direct involvement in DSB resection, the MRN complex plays a role in DSB sensing and activation of ATM (Difilippantonio and Nussenzweig, 2007). Interestingly, ATM kinase activity is also required for efficient DSB resection and subsequent activation of ATR (Adams et al., 2006;Cuadrado et al., 2006;Jazayeri et al., 2006;Myers and Cortez, 2006). These findings indicate that MRN and ATM promote the transition from DSB sensing to DNA end processing, although their exact role in this transition is not clear. In this report we have investigated the role of CtIP in the DSB damage.