Radiation-induced double-strand breaks require ATM but not Artemis for homologous recombination during S-phase

Abstract

Double-strand breaks (DSBs) are repaired by two distinct pathways, non-homologous end joining (NHEJ) and homologous recombination (HR). The endonuclease Artemis and the PIK kinase Ataxia-Telangiectasia Mutated (ATM), mutated in prominent human radiosensitivity syndromes, are essential for repairing a subset of DSBs via NHEJ in G1 and HR in G2. Both proteins have been implicated in DNA end resection, a mandatory step preceding homology search and strand pairing in HR. Here, we show that during S-phase Artemis but not ATM is dispensable for HR of radiation-induced DSBs. In replicating AT cells, numerous Rad51 foci form gradually, indicating a Rad51 recruitment process that is independent of ATM-mediated end resection. Those DSBs decorated with Rad51 persisted through S- and G2-phase indicating incomplete HR resulting in unrepaired DSBs and a pronounced G2 arrest. We demonstrate that in AT cells loading of Rad51 depends on functional ATR/Chk1. The ATR-dependent checkpoint response is most likely activated when the replication fork encounters radiation-induced single-strand breaks leading to generation of long stretches of single-stranded DNA. Together, these results provide new insight into the role of ATM for initiation and completion of HR during S- and G2-phase. The DSB repair defect during S-phase significantly contributes to the radiosensitivity of AT cells.

Increased oxidative stress in AOA3 cells disturbs ATM-dependent DNA damage responses

“…4C). The results of several studies suggest that ATM plays a role in the HR pathway [38][39][40]. The ATM-dependent phosphorylation of factors such as MDC1 and H2AX is important for the H2A/H2AX ubiquitination pathway and contributes to recruitment of several HR factors [35,36].…”

Profiling of the Chromatin-associated Proteome Identifies HP1BP3 as a Novel Regulator of Cell Cycle Progression

“…DNA-dependent protein kinase (DNA-PK) plays a key role in NHEJ-mediated repair of DSBs, and is comprised of a DNAbinding heterodimer Ku70/Ku80 and a catalytic subunit (DNAPKcs) that are necessary for the kinase activity of the enzyme (45). Euchromatin DSBs are repaired by NHEJ during G 1 and G 2 phase, whereas heterochromatin DSB repair is mediated by HR during G 2 phase (46,47), and perhaps also during S phase (48). Here we identified DSB repair proteins in "cluster B", our iTRAQ-based proteomic data and Western blot analysis demonstrated that Ku70 and Ku80 were substantially down-regulated in the pellet fractions (Fig.…”

Ataxia Telangiectasia Mutated (ATM) Is Dispensable for Endonuclease I-SceI-induced Homologous Recombination in Mouse Embryonic Stem Cells

“…3) Small molecule inhibitors of ATM and siRNA-mediated ATM depletion reduce HR in human cells (21,22). 4) In proliferating cells, although the majority (ϳ85%) of IR-induced DSBs are likely repaired by NHEJ with fast kinetics in an ATM-independent manner, repair of the remaining ϳ15% of IR-induced DSBs is dependent upon ATM and has slower repair kinetics that may reflect either NHEJ-mediated repair in heterochromatin or a possible HR-directed postreplication repair process (23)(24)(25)(26).…”