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

Abstract

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.

Fig. 1. Targeted recombination at the CHO APRT locus. The pAG6 and pAG6ins0.9 targeting vectors carry a full-length Chinese hamster APRT gene, disrupted by insertion of 33 or 945 bp of heterologous sequence into the exon 3 XhoI site. Linearization of pAG6 at a unique HindIII site within the heterologous insert creates an ends-in insertion vector configuration in which 18 and 11 terminal non-homologies flank each side of the DSB. Linearization of pAG6ins0.9 at a unique SalI site within the larger heterologous insert generates an insertion vector in which 271 and 670 nt terminal non-homologies flank each side of the DSB. The ERCC1⁺ (ATS49tg) and ERCC1^– mutant (U9S50tg) or knockout (E1KO7-5) cell lines used in this study all have the same 3 bp deletion in exon 5 of the APRT target gene locus, resulting in loss of the exon 5 MboII (Mb*) site. Electroporation of these cell lines with the pAG6 or pAG6ins0.9 targeting vectors, followed by selection for ALASA-resistant clones, results in recovery of APRT⁺ recombinants with the recombinant class structures shown. B, BamHI; Bg, BglII; E, EcoRI; H, HindIII; Mb, MboII; P, PstI; S, SacI; X, XhoI; Xb, XbaI.

Fig. 2. Southern analysis of APRT⁺ recombinants obtained from ATS49tg pAG6ins0.9 targeting experiments. Lane 1, APRT⁺ CHO-AT3-2 cells; lane 2, APRT^– ATS49tg cells; lane 3, a target gene conversion; lane 4, an MboII+/Δ targeted insertion; lane 5, an MboII+/+ targeted insertion; lane 6, a targeting vector correction. (A) MboII digests, 1.4 kb EcoRI–XbaI APRT probe. (B) HindIII–SacI double digests, 3.9 kb BamHI APRT probe. (C) BglII–XhoI double digests, 3.9 kb APRT probe.

Fig. 3. Southern analysis of aberrant insertion recombinants obtained from ERCC1^– cell pAG6ins0.9 targeting experiments. Lane 1, APRT^– E1KO7-5 cells; lanes 2–4, one normal targeted insertion (9U50/6ins S-17) and two aberrant insertion recombinants (9U50/6ins S-23, 9U50/6ins S-3) from U9S50tg targeting experiments; lanes 5–7, three aberrant insertion recombinants (99.12-14, 99.12-39 and 96.6.2-11) from E1KO7-5 targeting experiments. (A) MboII digests, 1.4 kb EcoRI–XbaI APRT probe. (B) PstI digests, 3.9 kb BamHI APRT probe. (C) BglII–XhoI double digests, 3.9 kb BamHI APRT probe. (D) HindIII–EcoRI double digests, 3.9 kb BamHI APRT probe.

Fig. 4. Structures of normal and aberrant targeted insertion recombinants. Deduced structures of the six APRT⁺ recombinants shown in Figure 3. Abbreviations as in Figure 1.