Nonhomologous End Joining during Restriction Enzyme-Mediated DNA Integration in Saccharomyces cerevisiae

Abstract

ABSTRACT The Bam HI restriction enzyme mediates integration of nonhomologous DNA into the Saccharomyces cerevisiae genome (R. H. Schiestl and T. D. Petes, Proc. Natl. Acad. Sci. USA 88:7585–7589, 1991). The present study investigates the mechanism of such events: in particular, the mediating activity of various restriction enzymes and the processing of resultant fragment ends. Our results show that in addition to Bam HI, Bgl II and Kpn I increase DNA integration efficiencies severalfold, while Asp 718, Hin dIII, Eco RI, Sal I, Sma I, Hpa I, Msc I, and Sna BI do not. Secondly, the three active enzymes stimulated integrations only of fragments containing 5′ or 3′ overhangs but not of blunt-ended fragments. Thirdly, integrations mediated by one enzyme and utilizing a substrate created by another required at least 2 bp of homology. Furthermore, an Asp 718 fragment possessing a 5′ overhang integrated into a Kpn I (isoschizomer) site possessing a 3′ overhang, most likely by filling of the 5′ overhang followed by 5′ exonuclease digestion to produce a 3′ end. We classified and analyzed the restriction enzyme-mediated integration events in the context of their genomic positions. The majority of events integrated into single sites. In the remaining 6 of 19 cases each end of the plasmid inserted into a different sequence, producing rearrangements such as duplications, deletions, and translocations.