Role of the nucleotide excision repair gene ERCC1 in formation of recombination-dependent rearrangements in mammalian cells.
Spontaneous recombination between direct repeats at the adenine phosphoribosyltransferase (APRT) locus in ERCC1-deficient cells generates a high frequency of rearrangements that are dependent on the process of homologous recombination, suggesting that rearrangements are formed by misprocessing of recombination intermediates. Given the specificity of the structure-specific Ercc1/Xpf endonuclease, two potential recombination intermediates are substrates for misprocessing in ERCC1(-) cells: heteroduplex loops and heteroduplex intermediates with non-homologous 3' tails. To investigate the roles of each, we constructed repeats that would yield no heteroduplex loops during spontaneous recombination or that would yield two non-homologous 3' tails after treatment with the rare-cutting endonuclease I-SCE:I. Our results indicate that misprocessing of heteroduplex loops is not the major source of recombination-dependent rearrangements in ERCC1-deficient cells. Our results also suggest that the Ercc1/Xpf endonuclease is required for efficient removal of non-homologous 3' tails, like its Rad1/Rad10 counterpart in yeast. Thus, it is likely that misprocessing of non-homologous 3' tails is the primary source of recombination-dependent rearrangements in mammalian cells. We also find an unexpected effect of ERCC1 deficiency on I-SCE:I-stimulated rearrangements, which are not dependent on homologous recombination, suggesting that the ERCC1 gene product may play a role in generating the rearrangements that arise after I-SCE:I-induced double-strand breaks.
Sargent
R G
RG
The Verna and Marrs McLean Department of Biochemistry and Molecular Biology, Baylor College of Medicine, 1 Baylor Plaza, Houston, TX 77030, USA.
Meservy
J L
JL
Perkins
B D
BD
Kilburn
A E
AE
Intody
Z
Z
Adair
G M
GM
Nairn
R S
RS
Wilson
J H
JH
D000228:Adenine Phosphoribosyltransferase / Q000235:genetics; D000818:Animals; D015139:Blotting, Southern; D002460:Cell Line; D003434:Crossing Over, Genetic / Q000235:genetics; D004247:DNA / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D004249:DNA Damage / Q000235:genetics; D004260:DNA Repair / Q000235:genetics*; D004268:DNA-Binding Proteins; D004720:Endonucleases; D017353:Gene Deletion; D009690:Nucleic Acid Conformation; D009692:Nucleic Acid Heteroduplexes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011506:Proteins / Q000235:genetics / Q000378:metabolism*; D011995:Recombination, Genetic / Q000235:genetics*; D012091:Repetitive Sequences, Nucleic Acid / Q000235:genetics; D013379:Substrate Specificity; D014162:Transfection
D004268:DNA-Binding Proteins; D009692:Nucleic Acid Heteroduplexes; D011506:Proteins; D004247:DNA; D000228:Adenine Phosphoribosyltransferase; D004720:Endonucleases