The effects of ethidium bromide and Mg++ ion on strand exchange in the Hin-mediated inversion reaction.
The biochemical reaction of a site-specific recombinase such as Hin invertase or gammadelta resolvase starts with binding of the recombinase to its recombination site and cleavage of the DNA in the center of the site. This is followed by strand exchange and finally ligation of the ends of the recombined strands. Previous biochemical studies have shown that Hin invertase and gammadelta resolvase cannot proceed beyond DNA cleavage in the absence of Mg++ ion, indicating that these recombinases require Mg++ ion in the strand exchange process. We have observed that the intercalating agent, ethidium bromide (2 microM), does not interfere with DNA cleavage, but slows strand exchange in a concentration-dependent manner. Levels of Mg++ ion below 5 mM also slow strand exchange substantially. We infer that random intercalation of ethidium bromide inhibits unwinding of the double helix at the recombination site in the negatively supercoiled DNA and propose that Mg+ may be required for Hin to deform the secondary structure of B-DNA prior to strand exchange.
Lee
Hee Jung
HJ
Department of Biology, College of Natural Sciences, Chungnam National University, Daejeon 305-764, Korea.
Lee
Young Lang
YL
Ji
Jeong Jun
JJ
Lim
Heon Man
HM
D007446:Chromosome Inversion; D004247:DNA / Q000378:metabolism; D004254:DNA Nucleotidyltransferases / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D004996:Ethidium / Q000378:metabolism*; D008274:Magnesium / Q000378:metabolism*; D009154:Mutation
D004247:DNA; D004254:DNA Nucleotidyltransferases; C059997:Hin recombinase; D004996:Ethidium; D008274:Magnesium