ERCC1-XPF endonuclease facilitates DNA double-strand break repair.
ERCC1-XPF endonuclease is required for nucleotide excision repair (NER) of helix-distorting DNA lesions. However, mutations in ERCC1 or XPF in humans or mice cause a more severe phenotype than absence of NER, prompting a search for novel repair activities of the nuclease. In Saccharomyces cerevisiae, orthologs of ERCC1-XPF (Rad10-Rad1) participate in the repair of double-strand breaks (DSBs). Rad10-Rad1 contributes to two error-prone DSB repair pathways: microhomology-mediated end joining (a Ku86-independent mechanism) and single-strand annealing. To determine if ERCC1-XPF participates in DSB repair in mammals, mutant cells and mice were screened for sensitivity to gamma irradiation. ERCC1-XPF-deficient fibroblasts were hypersensitive to gamma irradiation, and gammaH2AX foci, a marker of DSBs, persisted in irradiated mutant cells, consistent with a defect in DSB repair. Mutant mice were also hypersensitive to irradiation, establishing an essential role for ERCC1-XPF in protecting against DSBs in vivo. Mice defective in both ERCC1-XPF and Ku86 were not viable. However, Ercc1(-/-) Ku86(-/-) fibroblasts were hypersensitive to gamma irradiation compared to single mutants and accumulated significantly greater chromosomal aberrations. Finally, in vitro repair of DSBs with 3' overhangs led to large deletions in the absence of ERCC1-XPF. These data support the conclusion that, as in yeast, ERCC1-XPF facilitates DSB repair via an end-joining mechanism that is Ku86 independent.
University of Pittsburgh Cancer Institute, Hillman Cancer Center, Research Pavilion 2.6, 5117 Centre Avenue, Pittsburgh, Pennsylvania 15213-1863, USA.
Anwaar
A
Ahmad
Andria Rasile
AR
Robinson
Anette
A
Duensing
Ellen
E
van Drunen
H Berna
HB
Beverloo
David B
DB
Weisberg
Paul
P
Hasty
Jan H J
JH
Hoeijmakers
Laura J
LJ
Niedernhofer
D000818:Animals; D034961:Antigens, Nuclear / Q000378:metabolism; D002461:Cell Line, Transformed; D002470:Cell Survival / Q000528:radiation effects; D016922:Cellular Senescence / Q000528:radiation effects; D002869:Chromosome Aberrations / Q000528:radiation effects; D053903:DNA Breaks, Double-Stranded / Q000528:radiation effects; D004260:DNA Repair / Q000528:radiation effects; D004268:DNA-Binding Proteins / Q000172:deficiency / Q000378:metabolism*; D020964:Embryo Loss / Q000378:metabolism; D004622:Embryo, Mammalian / Q000166:cytology; D004720:Endonucleases / Q000172:deficiency / Q000378:metabolism*; D005347:Fibroblasts / Q000201:enzymology / Q000528:radiation effects; D042822:Genomic Instability / Q000528:radiation effects; D006367:HeLa Cells; D006657:Histones / Q000378:metabolism; D006801:Humans; D000072200:Ku Autoantigen; D051379:Mice; D010957:Plasmids / Q000235:genetics; D011839:Radiation, Ionizing; D017422:Sequence Analysis, DNA
D034961:Antigens, Nuclear; D004268:DNA-Binding Proteins; D006657:Histones; C475961:gamma-H2AX protein, mouse; C095888:xeroderma pigmentosum group F protein; D004720:Endonucleases; C499360:Ercc1 protein, mouse; C000606512:Xrcc5 protein, mouse; C000606509:Xrcc6 protein, human; C000606510:Xrcc6 protein, mouse; D000072200:Ku Autoantigen