The XPA-binding domain of ERCC1 is required for nucleotide excision repair but not other DNA repair pathways.
The endonuclease ERCC1-XPF incises the damaged strand of DNA 5' to a lesion during nucleotide excision repair (NER) and has additional, poorly characterized functions in interstrand cross-link repair, double-strand break repair, and homologous recombination. XPA, another key factor in NER, interacts with ERCC1 and recruits it to sites of damage. We identified ERCC1 residues that are critical for the interaction with XPA and assessed their importance for NER in vitro and in vivo. Mutation of two conserved residues (Asn-110 and Tyr-145) located in the XPA-binding site of ERCC1 dramatically affected NER but not nuclease activity on model DNA substrates. In ERCC1-deficient cells expressing ERCC1(N110A/Y145A), the nuclease was not recruited to sites of UV damage. The repair of UV-induced (6-4)photoproducts was severely impaired in these cells, and they were hypersensitive to UV irradiation. Remarkably, the ERCC1(N110A/Y145A) protein rescues the sensitivity of ERCC1-deficient cells to cross-linking agents. Our studies suggest that ERCC1-XPF engages in different repair pathways through specific protein-protein interactions and that these functions can be separated through the selective disruption of these interactions. We discuss the impact of these findings for understanding how ERCC1 contributes to resistance of tumor cells to therapeutic agents such as cisplatin.
Orelli
Barbara
B
From the Department of Pharmacological Sciences and Chemistry, Stony Brook University, Stony Brook, New York 11794-3400.
McClendon
T Brooke
TB
the Department of Microbiology and Molecular Genetics and Cancer Institute, Hillman Cancer Center, University of Pittsburgh, Pittsburgh, Pennsylvania 15213-1863.
Tsodikov
Oleg V
OV
the Department of Medicinal Chemistry, College of Pharmacy, University of Michigan, Ann Arbor, Michigan 48109-2676, and.
Ellenberger
Tom
T
the Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine, St. Louis, Missouri 63110.
Niedernhofer
Laura J
LJ
the Department of Microbiology and Molecular Genetics and Cancer Institute, Hillman Cancer Center, University of Pittsburgh, Pittsburgh, Pennsylvania 15213-1863.
Schärer
Orlando D
OD
From the Department of Pharmacological Sciences and Chemistry, Stony Brook University, Stony Brook, New York 11794-3400. Electronic address: orlando@pharm.stonybrook.edu.
D000595:Amino Acid Sequence; D000818:Animals; D001216:Asparagine / Q000235:genetics / Q000378:metabolism; D001665:Binding Sites / Q000235:genetics; D016466:CHO Cells; D002460:Cell Line; D002478:Cells, Cultured; D006224:Cricetinae; D003412:Cricetulus; D004249:DNA Damage; D004260:DNA Repair / Q000235:genetics / Q000502:physiology*; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004720:Endonucleases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D017434:Protein Structure, Tertiary; D017386:Sequence Homology, Amino Acid; D015398:Signal Transduction; D018411:Spodoptera; D014162:Transfection; D014443:Tyrosine / Q000235:genetics / Q000378:metabolism; D014466:Ultraviolet Rays; D051760:Xeroderma Pigmentosum Group A Protein / Q000235:genetics / Q000378:metabolism*
D004268:DNA-Binding Proteins; C497314:XPA protein, human; D051760:Xeroderma Pigmentosum Group A Protein; D014443:Tyrosine; D001216:Asparagine; C071447:ERCC1 protein, human; D004720:Endonucleases