Functional analysis of Box II mutations in yeast site-specific recombinases Flp and R. Significance of amino acid conservation within the Int family and the yeast sub-family.
The site-specific recombinases Flp and R from Saccharomyces cerevisiae and Zygosaccharomyces rouxii, respectively, are related proteins that share approximately 30% amino acid matches. They exhibit a common reaction mechanism that appears to be conserved within the larger Integrase family of site-specific recombinases. Two regions of the proteins, designated as Box I and Box II, harbor, in addition to amino acid conservation, a significantly high degree of nucleotide sequence homology within their coding segments. Box II also contains two amino acids, a histidine and an arginine, that are invariant throughout the Int family. We have performed functional analysis of Flp and R variants carrying point mutations within the Box II segment. Several positions within Box II can tolerate substitutions with no effect, or only modest effects on recombination. Alterations of the Int family residues, His305 and Arg308, in the R protein lead to the arrest of recombination at the strand cleavage or the strand exchange step. This is very similar to previously observed "step-arrest" phenotypes in Flp variants altered at these positions and has strong implications for the catalytic mechanism of recombination. Flp and R variants at His305 and His309 can be complemented in half-site strand transfer by a corresponding Tyr343 to phenylalanine variant. In contrast to Arg308 Flp variants, which are efficiently complemented in half-site strand transfer by Flp(Y343F), no strong complementation has been observed between Arg308 variants of R and R (Y343F).
Lee
J
J
Department of Microbiology, University of Texas, Austin 78712.
Serre
M C
MC
Yang
S H
SH
Whang
I
I
Araki
H
H
Oshima
Y
Y
Jayaram
M
M
D004252:DNA Mutational Analysis; D004254:DNA Nucleotidyltransferases / Q000235:genetics* / Q000378:metabolism; D005816:Genetic Complementation Test; D019426:Integrases; D008957:Models, Genetic; D016297:Mutagenesis, Site-Directed; D045522:Recombinases; D011995:Recombination, Genetic; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics; D017386:Sequence Homology, Amino Acid; D012689:Sequence Homology, Nucleic Acid; D013329:Structure-Activity Relationship; D015003:Yeasts / Q000235:genetics* / Q000378:metabolism
D045522:Recombinases; D004254:DNA Nucleotidyltransferases; D019426:Integrases; C115006:integron integrase IntI1