DNA recombination and RNA cleavage activities of the Flp protein: roles of two histidine residues in the orientation and activation of the nucleophile for strand cleavage.
Using a combination of DNA and hybrid DNA-RNA substrates, we have analyzed the mechanism of phosphoryl transfer by the Flp site-specific recombinase in three different reactions: DNA strand breakage and joining, and two types of RNA cleavage activities. These reactions were then used to characterize Flp variants altered at His309 and His345, amino acid residues that are in close proximity to two key catalytic residues (Arg308 and Tyr343). These histidine residues are important for strand cutting by Tyr343, the active-site nucleophile of Flp, but neither residue contributes to the type II RNA cleavage activity or to the strand-joining reaction in a pre-cleaved substrate. Strand cleavage reactions using small, diffusible nucleophiles indicate that this histidine pair contributes to the correct positioning and activation of Tyr343 within the shared active site of Flp. The implications of these results are evaluated against the recently solved crystal structure of Flp in association with a Holliday junction.
Grainge
I
I
Section of Molecular Genetics and Microbiology, University of Texas at Austin, Austin, TX 78712, USA.
Lee
J
J
Xu
C J
CJ
Jayaram
M
M
D019943:Amino Acid Substitution / Q000235:genetics; D001665:Binding Sites; D002384:Catalysis; D004247:DNA / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D004254:DNA Nucleotidyltransferases / Q000737:chemistry* / Q000145:classification / Q000235:genetics / Q000378:metabolism*; D005816:Genetic Complementation Test; D006639:Histidine / Q000235:genetics / Q000378:metabolism*; D006861:Hydrogen Peroxide / Q000378:metabolism; D009690:Nucleic Acid Conformation; D009692:Nucleic Acid Heteroduplexes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017354:Point Mutation / Q000235:genetics; D012313:RNA / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011995:Recombination, Genetic / Q000235:genetics*; D012260:Ribonucleases / Q000737:chemistry / Q000145:classification / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D014439:Tyramine / Q000378:metabolism
D009692:Nucleic Acid Heteroduplexes; D006639:Histidine; D012313:RNA; D004247:DNA; D006861:Hydrogen Peroxide; D004254:DNA Nucleotidyltransferases; C045119:FLP recombinase; D012260:Ribonucleases; D014439:Tyramine