Effects of dimer interface mutations in Hin recombinase on DNA binding and recombination.
Previous biochemical assays and a structural model of the protein have indicated that the dimer interface of the Hin recombinase is composed of two alpha-helices. To elucidate the structure and function of the helix, amino acids at the N-terminal end of the helix, where the two helices make their most extensive contact, were randomized, and inversion-incompetent mutants were selected. To investigate why the mutants lost their inversion activities, the DNA binding, hix pairing, invertasome formation, and DNA cleavage activities were assayed using in vivo and in vitro methodologies. The results indicated that the mutants could be divided into four classes based on their DNA binding activity. We propose that the alpha-helices might serve to place a DNA binding motif of Hin in the correct spatial relationship to the minor groove of the recombination site. All the mutants except those that failed to bind DNA were able to perform hix pairing and invertasome formation, suggesting that the dimer interface is not involved in either of these processes. The inversion-incompetent phenotype of the binders was caused by the inability of mutants to perform DNA cleavage. The mutants that showed less binding ability than the wild type nevertheless exhibited a wild-type level of hix pairing activity, because the hix pairing activity overcomes the defect in DNA binding. This phenotype of the mutants that are impaired in DNA binding suggests that the binding domains of Hin may mediate Hin-Hin interaction during hix pairing.
Lee
H J
HJ
Department of Biology, College of Natural Sciences, Chungnam National University, 220 Kung-Dong, Taejon 305-764, Republic of Korea.
Lee
S Y
SY
Lee
H
H
Lim
H M
HM
D001426:Bacterial Proteins / Q000235:genetics* / Q000378:metabolism; D001665:Binding Sites; D004254:DNA Nucleotidyltransferases / Q000235:genetics* / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000235:genetics* / Q000378:metabolism; D019281:Dimerization; D008957:Models, Genetic; D008958:Models, Molecular; D009154:Mutation; D010957:Plasmids / Q000378:metabolism; D011485:Protein Binding; D011995:Recombination, Genetic / Q000235:genetics*; D013499:Surface Properties
D001426:Bacterial Proteins; D004268:DNA-Binding Proteins; D004254:DNA Nucleotidyltransferases; C059997:Hin recombinase