Multistep signaling of DNA double-stranded breaks. ATM and ATR, through the phosphorylation of their downstream substrates, play central roles in DSB signaling and the activation of cell cycle checkpoints. (A) Mre11/Rad50/NBS1 (MRN)-complex mainly recognizes DSBs and undergo ATM activation. Activated ATM phophorylates multiple substrates, including H2A.X and MDC1. MDC1 recognizes γ-H2A.X and bind to it. Then RNF8 recognizes the thyreonyl-phosphorylated MDC1 via its FHA domain and is recruited to DSB sites where it ubiquitylates the histone H2A. Subsequently, RNF168 is recruited to the ubiquitylated H2A, further ubiquitylating it with Ubc13 and likely leading to the modification of the chromatin structure. Through its tutor domain, 53BP1 is recruited to histone H4 di-methylated on lysine 20, while RAP80 and its protein complex are recruited to the polyubiquitylated H2A. (B) In the S and G2 phase, DSBs are recognized by the MRN-complex and CtIP is recruited to DSBs. CtIP is phosphorylated by CDK and ATM, and is ubiquitylated by BRCA1 in response to DNA damage. Collaboration of MRN and CtIP results in DSB end resection, and subsequently DNA2/BLM-complex and Exo1 promote further the resection of DSB ends leading to generation of single strand DNA (ssDNA). RPA binds to the ssDNA and induces ATR activation and HR repair [181–184]. (C) Replication fork stalling. The damaged single-stranded DNA is coated by RPA and this structure plays crucial roles in the recruitment of the helicase HARP and the ATR-ATRIP complex to sites of DNA damage[185–187]. P: phosphorylation, Ub: ubiquitin, S: SUMOylation, ATRIP: ATR interacting protein, RPA: Replication protein A. HARP: HepA-related protein.