Studies on the response to DNA double-strand breaks: Damage-dependent phosphorylations and DNA end resection

DNA double-strand breaks (DSBs) are the most severe form of DNA damage, since if not properly repaired they can lead to loss of heterozygosity, chromosomal translocation, and even chromosomal fragmentation and loss. ATM and ATR are the two principal upstream kinases which respond to DNA damage and activate DNA damage responses. ATM responds directly to DNA double-strand breaks (DSBs), while ATR responds to primed single-stranded DNA (ssDNA). Eukaryotic cells have two pathways to repair DSBs: non-homologous end joining (NHEJ) and homology-directed repair (HDR). NHEJ repair is error-prone and can lead to promiscuous chromosome fusions if multiple DSBs are present in the same cell. HDR pathways are high-fidelity since they utilize sequence homology elsewhere in the genome to facilitate recovery of the lost sequence and repair of the break. HDR pathways are initiated by the degradation of the 5' strand of the DSB, yielding a 3' single-stranded DNA overhang. This process is termed DNA resection, and not only allows all subsequent steps of HDR, but also makes the DSB refractory to repair by the NHEJ pathway.;We have conducted an expression screen to identify proteins which are post-translationally modified in response to DNA DSBs, in an ATM/ATR-dependent manner. This screen has yielded several proteins of diverse and unexpected function which respond to DSBs.;Additionally, we used Xenopus laevis cell-free egg extracts in S-phase or M-phase to monitor recruitment of signaling and repair proteins at chromosomal DSBs and to assess DNA end resection. While MRN is required for both resection and ATM signaling from DSBs, CtIP acts downstream of the MRN complex for resection, and is dispensable for ATM signaling. In S-phase, the MRN-CtIP pathway acts early in DSB processing followed by MRN-CtIP-independent end resection. Notably, DNA end resection is exclusively dependent on the CtIP-MRN pathway in M-phase. MRN-CtIP-independent resection in S-phase does not require DNA synthesis or ATM activity. However, Cdk1 activity plays a critical role in DNA damage signaling in M-phase. We establish that the BRCA1 protein is not required for DSB resection. Finally, we demonstrate that early binding of NHEJ factors and HDR factors occurs non-competitively.