Microhomology-mediated end joining (MMEJ) is a major pathway for Ku-independent alternative nonhomologous end joining, which contributes to chromosomal translocations and telomere fusions, but the underlying mechanism of MMEJ in mammalian cells is not well understood. In this study, we demonstrated that, distinct from Ku-dependent classical nonhomologous end joining, MMEJ-even with very limited end resection-requires cyclin-dependent kinase activities and increases significantly when cells enter S phase. We also showed that MMEJ shares the initial end resection step with homologous recombination (HR) by requiring meiotic recombination 11 homolog A (Mre11) nuclease activity, which is needed for subsequent recruitment of Bloom syndrome protein (BLM) and exonuclease 1 (Exo1) to DNA double-strand breaks (DSBs) to promote extended end resection and HR. MMEJ does not require S139-phosphorylated histone H2AX (γ-H2AX), suggesting that initial end resection likely occurs at DSB ends. Using a MMEJ and HR competition repair substrate, we demonstrated that MMEJ with short end resection is used in mammalian cells at the level of 10-20% of HR when both HR and nonhomologous end joining are available. Furthermore, MMEJ is used to repair DSBs generated at collapsed replication forks. These studies suggest that MMEJ not only is a backup repair pathway in mammalian cells, but also has important physiological roles in repairing DSBs to maintain cell viability, especially under genomic stress.BLM/Exo1 | CtIP | DNA repair pathway | DNA damage | genome stability D NA double-strand breaks (DSBs) can be repaired by multiple pathways. The classical nonhomologous end joining (C-NHEJ) pathway relies on Ku70/Ku80 and ligates DSB ends without a template (1). Homologous recombination (HR), an error-free pathway, uses a homologous template to repair DSBs (2) and is initiated by end resection from DSB ends to generate a long stretch of singlestrand DNA (ssDNA) for strand invasion. Although C-NHEJ is active throughout the cell cycle, HR is used when cells enter S and G2 because cyclin-dependent kinases (CDKs) are needed for promoting end resection to activate HR (3-5).In the absence of C-NHEJ factors such as Ku70, Ku80, or DNA ligase IV, robust alternative nonhomologous end joining (alt-NHEJ) activity is observed in various organisms including yeast and mammals (6, 7). Many alt-NHEJ events, classified as microhomology-mediated end joining (MMEJ), require end resection and join the ends by base pairing at microhomology sequences (5-25 nucleotides), resulting in deletions at the junctions (6). However, other alt-NHEJ pathways without using microhomology regions also exist.Genetic analyses in yeast reveal that MMEJ is Rad52-independent, distinguishing it from HR and single-strand annealing (SSA) repair pathways, whereas the Mre11-Rad50-Xrs2 (MRX) complex and DNA ligase IV are needed for MMEJ (8-10). Further studies suggest that in yeast, Srs2 helicase, Sae2 nuclease [CtBP-interacting protein (CtIP) homologue], Tel1 [ataxia telangiectasia mutated (A...

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.

CtIP plays an important role in homologous recombination (HR)–mediated DNA double-stranded break (DSB) repair and interacts with Nbs1 and BRCA1, which are linked to Nijmegen breakage syndrome (NBS) and familial breast cancer, respectively. We identified new CDK phosphorylation sites on CtIP and found that phosphorylation of these newly identified CDK sites induces association of CtIP with the N-terminus FHA and BRCT domains of Nbs1. We further showed that these CDK-dependent phosphorylation events are a prerequisite for ATM to phosphorylate CtIP upon DNA damage, which is important for end resection to activate HR by promoting recruitment of BLM and Exo1 to DSBs. Most notably, this CDK-dependent CtIP and Nbs1 interaction facilitates ATM to phosphorylate CtIP in a substrate-specific manner. These studies reveal one important mechanism to regulate cell-cycle-dependent activation of HR upon DNA damage by coupling CDK- and ATM-mediated phosphorylation of CtIP through modulating the interaction of CtIP with Nbs1, which significantly helps to understand how DSB repair is regulated in mammalian cells to maintain genome stability.

Proper recognition and repair of DNA damage is critical for the cell to protect its genomic integrity. Laser microirradiation ranging in wavelength from ultraviolet A (UVA) to near-infrared (NIR) can be used to induce damage in a defined region in the cell nucleus, representing an innovative technology to effectively analyze the in vivo DNA double-strand break (DSB) damage recognition process in mammalian cells. However, the damage-inducing characteristics of the different laser systems have not been fully investigated. Here we compare the nanosecond nitrogen 337 nm UVA laser with and without bromodeoxyuridine (BrdU), the nanosecond and picosecond 532 nm green second-harmonic Nd:YAG, and the femtosecond NIR 800 nm Ti:sapphire laser with regard to the type(s) of damage and corresponding cellular responses. Crosslinking damage (without significant nucleotide excision repair factor recruitment) and single-strand breaks (with corresponding repair factor recruitment) were common among all three wavelengths. Interestingly, UVA without BrdU uniquely produced base damage and aberrant DSB responses. Furthermore, the total energy required for the threshold H2AX phosphorylation induction was found to vary between the individual laser systems. The results indicate the involvement of different damage mechanisms dictated by wavelength and pulse duration. The advantages and disadvantages of each system are discussed.

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