Nonhomologous End Joining during Restriction Enzyme-Mediated DNA Integration in Saccharomyces cerevisiae
AUTOR(ES)
Manivasakam, Palaniyandi
FONTE
American Society for Microbiology
RESUMO
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.
ACESSO AO ARTIGO
http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=108888Documentos Relacionados
- Tagging developmental genes in Dictyostelium by restriction enzyme-mediated integration of plasmid DNA.
- Effect of mutations in genes affecting homologous recombination on restriction enzyme-mediated and illegitimate recombination in Saccharomyces cerevisiae.
- Tagged mutations at the Tox1 locus of Cochliobolus heterostrophus by restriction enzyme-mediated integration.
- Deletions in the Gibberellin Biosynthesis Gene Cluster of Gibberella fujikuroi by Restriction Enzyme-Mediated Integration and Conventional Transformation-Mediated Mutagenesis
- Spectrophotometric assay for enzyme-mediated unwinding of double-stranded DNA.