Abstract:
:The BamHI 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 BamHI, BglII and KpnI increase DNA integration efficiencies severalfold, while Asp718, HindIII, EcoRI, SalI, SmaI, HpaI, MscI, and SnaBI 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 Asp718 fragment possessing a 5' overhang integrated into a KpnI (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.
journal_name
Mol Cell Bioljournal_title
Molecular and cellular biologyauthors
Manivasakam P,Schiestl RHdoi
10.1128/mcb.18.3.1736subject
Has Abstractpub_date
1998-03-01 00:00:00pages
1736-45issue
3eissn
0270-7306issn
1098-5549journal_volume
18pub_type
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