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PMID: 9488490 Published · ppublish English Journal Article Research Support, Non-U.S. Gov't

Nonhomologous end joining during restriction enzyme-mediated DNA integration in Saccharomyces cerevisiae.

Molecular and cellular biology ·Vol. 18 ·No. 3 ·1998-03-00 ·Pages 1736-45

Manivasakam P, Schiestl RH

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.

MeSH Terms
DNA, Single-Stranded Deoxyribonuclease BamHI/metabolism Deoxyribonucleases, Type II Site-Specific/metabolism Saccharomyces cerevisiae/genetics Transformation, Genetic
Chemicals
DNA, Single-Stranded Deoxyribonuclease BamHI Deoxyribonucleases, Type II Site-Specific GCCNNNNNGGC-specific type II deoxyribonucleases GGTACC-specific type II deoxyribonucleases
Authors & Affiliations
2 authors, click to expand affiliations / ORCID
Manivasakam P
Department of Molecular and Cellular Toxicology, Harvard School of Public Health, Boston, Massachusetts 02115, USA.
Schiestl R H
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Article Info
Journal
Molecular and cellular biology
Abbr.
Mol Cell Biol
ISSN
0270-7306
DOI
10.1128/MCB.18.3.1736
Published
1998-03-00
Pages
1736-45
Language
English
Region
United States
NLM ID
8109087
PMCID
PMC108888
Subset
IM
External Links
PubMed source DOI: 10.1128/MCB.18.3.1736 PMC Full Text View journal details
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