Role of ERCC1 in removal of long non-homologous tails during targeted homologous recombination.
The XpF/Ercc1 structure-specific endonuclease performs the 5' incision in nucleotide excision repair and is the apparent mammalian counterpart of the Rad1/Rad10 endonuclease from Saccharomyces cerevisiae. In yeast, Rad1/Rad10 endonuclease also functions in mitotic recombination. To determine whether XpF/Ercc1 endonuclease has a similar role in mitotic recombination, we targeted the APRT locus in Chinese hamster ovary ERCC1(+) and ERCC1(-) cell lines with insertion vectors having long or short terminal non-homologies flanking each side of a double-strand break. No substantial differences were evident in overall recombination frequencies, in contrast to results from targeting experiments in yeast. However, profound differences were observed in types of APRT(+) recombinants recovered from ERCC1(-) cells using targeting vectors with long terminal non-homologies-almost complete ablation of gap repair and single-reciprocal exchange events, and generation of a new class of aberrant insertion/deletion recombinants absent in ERCC1(+) cells. These results represent the first demonstration of a requirement for ERCC1 in targeted homologous recombination in mammalian cells, specifically in removal of long non-homologous tails from invading homologous strands.
Adair
G M
GM
Department of Carcinogenesis, The University of Texas M.D. Anderson Cancer Center, Science Park-Research Division, Smithville, TX 78957, USA.
Rolig
R L
RL
Moore-Faver
D
D
Zabelshansky
M
M
Wilson
J H
JH
Nairn
R S
RS
D000228:Adenine Phosphoribosyltransferase / Q000235:genetics / Q000378:metabolism; D000818:Animals; D015139:Blotting, Southern; D016466:CHO Cells; D002460:Cell Line; D006224:Cricetinae; D004247:DNA / Q000235:genetics / Q000378:metabolism; D004260:DNA Repair / Q000235:genetics*; D004268:DNA-Binding Proteins; D018274:Electroporation; D004720:Endonucleases; D017353:Gene Deletion; D018390:Gene Targeting; D005822:Genetic Vectors / Q000235:genetics; D016254:Mutagenesis, Insertional / Q000235:genetics; D011506:Proteins / Q000235:genetics / Q000378:metabolism*; D011995:Recombination, Genetic / Q000235:genetics*; D012689:Sequence Homology, Nucleic Acid
D004268:DNA-Binding Proteins; D011506:Proteins; D004247:DNA; D000228:Adenine Phosphoribosyltransferase; D004720:Endonucleases