Resolution and conservation of mismatches in DNA end joining.

Abstract

DNA end joining is a major pathway for the elimination of double-strand breaks from chromosomal DNA of higher eucaryotic cells. Extracts of Xenopus laevis eggs rejoin such breaks even when their short single-stranded termini are expected to form imperfectly matched overlaps. However, end-joined products cloned in Escherichia coli, necessarily give rise to perfectly matched products. Therefore it has not been possible to determine whether the end joining process creates mismatched products, perfectly matched (resolved) products or both. To investigate whether mismatch resolution was the result of the X. laevis end joining process or of activities of the bacterial host we used denaturing gradient gel electrophoresis to analyse joined products. We found that the end joining process does include mismatch resolution, the degree of which varies with regard to the nature of the original overlap structure. Mismatches 3' to a gap are completely resolved, mismatches 3' to a nick and 5' to a nick or gap are resolved to some extent but are generally conserved. Mismatches between base matches are always conserved. These findings suggest competing processes of ligation, DNA fill-in synthesis or exonucleolytic excision of mismatched bases next to a gap or nick. At mismatches 3' to a nick the probability of ligation is greater than that of excision while at mismatches 3' to a gap the probability of excision is greater than elongation of a given mismatch. At mismatches 5' to nicks or gaps it appears that ligation or elongation and ligation, respectively, are the most probable pathways but products resulting from mismatch excision, elongation and ligation are also detected.