FIG. 2.

DSB repair by allelic recombination and by NHEJ. (A) A DSB generated in the S2neo gene by the I-SceI endonuclease can be repaired by mechanisms that result in loss of the I-SceI site, as shown in the diagram. Repair of the DSB by allelic recombination, using the Pneo allele as a template for repair, results in replacement of the I-SceI site with an NcoI site and creation of a wild-type neo gene. Alternatively, repair of the DSB can occur by NHEJ, resulting in insertions (+) or deletions (Δ) at the I-SceI site. After the primers indicated by the arrows were used for PCR amplification of the S2neo gene, digestion of the PCR product by I-SceI and NcoI and resolution of the products on an agarose gel along with uncut product were performed and alterations to the I-SceI site as a consequence of DSB repair were analyzed. (B) In G418-resistant recombinants, the I-SceI site has been converted into an NcoI site. The PCR products of the rb1 and p6− primer set for the parental cell line (S/P) and a G418-resistant allelic recombinant arising from expression of I-SceI (R) are shown. S, I-SceI; N, NcoI; U, uncut product. (C) Quantification of total loss of the I-SceI site. The PCR products of the rb1 and p6+ primer set for the parental cell line prior to I-SceI transfection or on the 5th day of growth in nonselective media after I-SceI transfection are shown. The faint I-SceI-resistant fragment observed from the I-SceI transfected cells (but not from the S/P cells) is approximately 5% of the total amplification product.