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Molecular and Cellular Biology, August 2008, p. 5082-5092, Vol. 28, No. 16
0270-7306/08/$08.00+0     doi:10.1128/MCB.00293-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

ERCC1-XPF Endonuclease Facilitates DNA Double-Strand Break Repair ^,

University of Pittsburgh Cancer Institute, Hillman Cancer Center, Research Pavilion 2.6, 5117 Centre Avenue, Pittsburgh, Pennsylvania 15213-1863,¹ Department of Microbiology and Molecular Genetics, University of Pittsburgh School of Medicine, E1240 BSTWR, 200 Lothrop St., Pittsburgh, Pennsylvania 15261,² Department of Human Genetics, University of Pittsburgh School of Public Health, A300 Crabtree Hall, GSPH, 130 Desoto Street, Pittsburgh, Pennsylvania 15261,³ Department of Pathology, University of Pittsburgh School of Medicine, S417 BSTWR, 200 Lothrop St., Pittsburgh, Pennsylvania 15261,⁴ TumorCytogenetic Laboratory, Department of Clinical Genetics, Erasmus Medical Center, P.O. Box 1738, 3000 DR Rotterdam, The Netherlands,⁵ Department of Molecular Medicine, University of Texas Health Science Center, San Antonio, Texas 78245-3207,⁶ Medical Genetics Center, Department of Cell Biology and Genetics, Center of Biomedical Genetics, Erasmus Medical Center, P.O. Box 1738, 3000 DR Rotterdam, The Netherlands⁷

Received 21 February 2008/ Returned for modification 17 March 2008/ Accepted 29 May 2008

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 H2AX 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.

Published ahead of print on 9 June 2008.

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