The structure-specific endonuclease Ercc1-Xpf is required for targeted gene replacement in embryonic stem cells

The Ercc1-Xpf heterodimer, a highly conserved structure-specific endonuclea se, functions in multiple DNA repair pathways that are pivotal for maintain ing genome stability, including nucleotide excision repair, interstrand cro sslink repair and homologous recombination. Ercc1-Xpf incises double-strand ed DNA at double-strand/single-strand junctions, making it an ideal enzyme for processing DNA structures that contain partially unwound strands. Here we demonstrate that although Ercc1 is dispensable for recombination between sister chromatids, it is essential for targeted gene replacement in mouse embryonic stem cells. Surprisingly, the role of Ercc1-Xpf in gene targeting is distinct from its previously identified role in removing nonhomologous termini from recombination intermediates because it was required irrespecti ve of whether the ends of the DNA targeting constructs were heterologous or homologous to the genomic locus. Our observations have implications for th e mechanism of gene targeting in mammalian cells and define a new role for Ercc1-Xpf in mammalian homologous recombination. We propose a model for the mechanism of targeted gene replacement that invokes a role for Ercc1-Xpf i n making the recipient genomic locus receptive for gene replacement.