Role of ERCC1 in removal of long non-homologous tails during targeted homologous recombination

• Published in: The EMBO journal Vol. 19; no. 20; pp. 5552 - 5561
• Main Authors: Adair, Gerald M., Rolig, Rhonda L., Moore-Faver, Dana, Zabelshansky, Marina, Wilson, John H., Nairn, Rodney S.
• Format: Journal Article
• Language: English
• Published: Chichester, UK John Wiley & Sons, Ltd 16.10.2000; Blackwell Publishing Ltd; Oxford University Press
• Subjects: Adenine Phosphoribosyltransferase - genetics; Adenine Phosphoribosyltransferase - metabolism; Animals; APRT gene; Blotting, Southern; Cell Line; CHO Cells; Cricetinae; DNA - genetics; DNA - metabolism; DNA Repair - genetics; DNA-Binding Proteins; Electroporation; Endonucleases; ERCC1; ERCC1 gene; Ercc1 protein; Gene Deletion; Gene Targeting; Genetic Vectors - genetics; homologous recombination; Mammals; Mutagenesis, Insertional - genetics; Proteins - genetics; Proteins - metabolism; Rad1 protein; Rad10 protein; Recombination, Genetic - genetics; Saccharomyces cerevisiae; Sequence Homology, Nucleic Acid; terminal non-homology; XpF protein; XpF-Ercc1 endonuclease; Yeasts
• ISSN: 0261-4189; 1460-2075; 1460-2075
• DOI: 10.1093/emboj/19.20.5552

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.