ABSTRACT
Homologous recombination (HR) and non-homologous end joining (NHEJ) represent distinct pathways for repairing DNA double-strand breaks (DSBs). Previous work implicated Artemis and ATM in an NHEJ-dependent process, which repairs a defined subset of radiation-induced DSBs in G1-phase. Here, we show that in G2, as in G1, NHEJ represents the major DSB-repair pathway whereas HR is only essential for repair of approximately 15% of X- or gamma-ray-induced DSBs. In addition to requiring the known HR proteins, Brca2, Rad51 and Rad54, repair of radiation-induced DSBs by HR in G2 also involves Artemis and ATM suggesting that they promote NHEJ during G1 but HR during G2. The dependency for ATM for repair is relieved by depleting KAP-1, providing evidence that HR in G2 repairs heterochromatin-associated DSBs. Although not core HR proteins, ATM and Artemis are required for efficient formation of single-stranded DNA and Rad51 foci at radiation-induced DSBs in G2 with Artemis function requiring its endonuclease activity. We suggest that Artemis endonuclease removes lesions or secondary structures, which inhibit end resection and preclude the completion of HR or NHEJ.
Subject(s)
Cell Cycle Proteins/metabolism , DNA Breaks, Double-Stranded/radiation effects , DNA-Binding Proteins/metabolism , G2 Phase/radiation effects , Nuclear Proteins/metabolism , Protein Serine-Threonine Kinases/metabolism , Tumor Suppressor Proteins/metabolism , Apoptosis Regulatory Proteins , Ataxia Telangiectasia Mutated Proteins , BRCA2 Protein/metabolism , Cell Cycle Proteins/genetics , Cells, Cultured , DNA Helicases , DNA Repair/drug effects , DNA Repair Enzymes/metabolism , DNA-Binding Proteins/genetics , Endonucleases , Fibroblasts/radiation effects , G1 Phase/radiation effects , Gene Deletion , HeLa Cells , Heterochromatin/metabolism , Humans , Nuclear Proteins/genetics , Protein Serine-Threonine Kinases/genetics , Rad51 Recombinase/metabolism , Replication Protein A/metabolism , Tumor Suppressor Proteins/geneticsABSTRACT
DNA double-strand break (DSB) repair and checkpoint control represent distinct mechanisms to reduce chromosomal instability. Ataxia telangiectasia (A-T) cells have checkpoint arrest and DSB repair defects. We examine the efficiency and interplay of ATM's G2 checkpoint and repair functions. Artemis cells manifest a repair defect identical and epistatic to A-T but show proficient checkpoint responses. Only a few G2 cells enter mitosis within 4 h after irradiation with 1 Gy but manifest multiple chromosome breaks. Most checkpoint-proficient cells arrest at the G2/M checkpoint, with the length of arrest being dependent on the repair capacity. Strikingly, cells released from checkpoint arrest display one to two chromosome breaks. This represents a major contribution to chromosome breakage. The presence of chromosome breaks in cells released from checkpoint arrest suggests that release occurs before the completion of DSB repair. Strikingly, we show that checkpoint release occurs at a point when approximately three to four premature chromosome condensation breaks and approximately 20 gammaH2AX foci remain.
