FIG. 4.

Analysis of DSB-induced intrachromosomal recombination in wild-type and Msh2^−/− cell lines. (A) After in vivo expression of the I-SceI endonuclease, a DSB was introduced in the S2neo gene (top bar) of the H-DR-8mu substrate. Recombinational repair was initiated using the pneo-8mu template (bottom bar), restoring the NcoI site at the former position of the I-SceI site (thick hatch marks) to form a neo⁺ gene and, in this case, creating hDNA at the downstream NsiI and PmlI restriction site polymorphisms (thin hatch marks). Correction of the mismatched hDNA in a wild-type cell, followed by replication and division, results in both daughter cells containing the same genotype. However, in Msh2^−/− cells, MMR correction does not occur, and the uncorrected strands are segregated to daughter cells. (B) PCR analysis of two neo⁺ clones after DSB-induced recombination in the H-DR-8mu substrate. The neo⁺ gene coding region was amplified by PCR as an 810-bp fragment from genomic DNA with primers Neo1 and Neo3 and electrophoresed on agarose gels following digestion with the indicated restriction enzymes (Fig. 3). The amplified product from each of the neo⁺ clones was not cleaved by I-SceI (data not shown) but was cleaved by NcoI (Nco) and other restriction enzymes, as indicated by arrows. In the wild-type recombinant 3A-10, the NsiI (Ns) and PmlI (Pm) sites were fully cleaved, indicating a population of cells with one genotype (as in lane A). By contrast, in the Msh2^−/− recombinant 17A-9, both the NsiI and the PmlI sites were partially cleaved, indicating a population of cells with two genotypes (as in lane A). Note that due to the naturally occurring PstI (P) site, the amplified fragment in each of the clones was shifted when cleaved with PstI. M, mixed digest; MW, 1-kb ladder molecular weight marker; A, ApaI; L, ApaLI; B, BamHI; X, XbaI; Nr, NruI.