Role of arginine-43 and arginine-69 of the Hin recombinase catalytic domain in the binding of Hin to the hix DNA recombination sites.
The Hin recombinase mediates the site-specific inversion of a segment of the Salmonella chromosome between two flanking 26bp hix DNA recombination sites. Mutations in two amino acid residues, R43 and R69 of the catalytic domain of the Hin recombinase, were identified that can compensate for loss of binding resulting from elimination of certain major and minor groove contacts within the hix recombination sites. With one exception, the R43 and R69 mutants were also able to bind a hix sequence with an additional 4bp added to the centre of the site, unlike wild-type Hin. Purified Hin mutants R43H and R69C had both partial cleavage and inversion activities in vitro while mutants R43L, R43C, R69S, and R69P had no detectable cleavage and inversion activities. These data support a model in which the catalytic domain plays a role in DNA-binding specificity, and suggest that the arginine residues at positions 43 and 69 function to position the Hin recombinase on the DNA for a step in the recombination reaction which occurs either at and/or prior to DNA cleavage.
Adams
C W
CW
Department of Microbiology, University of Washington, Seattle 98195, USA. cervesa@u.washington.edu
Nanassy
O
O
Johnson
R C
RC
Hughes
K T
KT
D001120:Arginine / Q000378:metabolism*; D001665:Binding Sites; D002384:Catalysis; D007446:Chromosome Inversion; D004254:DNA Nucleotidyltransferases / Q000235:genetics / Q000378:metabolism*; D004269:DNA, Bacterial / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics; D016296:Mutagenesis; D011995:Recombination, Genetic; D012486:Salmonella typhimurium / Q000235:genetics; D012694:Serine / Q000378:metabolism; D013379:Substrate Specificity
D004269:DNA, Bacterial; D004268:DNA-Binding Proteins; D012694:Serine; D001120:Arginine; D004254:DNA Nucleotidyltransferases; C059997:Hin recombinase