Biochemical and kinetic analysis of the RNase active sites of the integrase/tyrosine family site-specific DNA recombinases.
In this study, we have used multiple strategies to characterize the mechanisms of the type I and type II RNA cleavage activities harbored by the Flp (pronounced here as "flip") site-specific DNA recombinase (Flp-RNase I and II, respectively). Reactions using half-sites pre-bound by step-arrest mutants of Flp agree with a "shared active site" being responsible for the type I reaction (as is the case with normal DNA recombination). In a "pre-cleaved" type I substrate containing a 3'-phosphotyrosyl bond, the Flp-RNase I activity can be elicited by either wild type Flp or by Flp(Y343F). Kinetic analyses of the type I reaction are consistent with the above observations and support the notion that the DNA recombinase and type I RNase active sites are identical. The type II RNase activity is expressed by Flp(Y343F) in a half-site substrate and is unaffected by the catalytic constitution of a Flp monomer present on a partner half-site. Reaction conditions that proscribe the assembly of a DNA bound Flp dimer have no effect on Flp-RNase II. These biochemical results, together with kinetic data, are consistent with the reaction being performed from a "non-shared active site" contained within a single Flp monomer. The Flp-related recombinase Cre, which utilizes a non-shared recombination active site, exhibits the type I RNA cleavage reaction. So far, we have failed to detect the type II RNase activity in Cre. Despite their differences in active site assembly, Cre functionally mimics Flp in being able to provide two functional active sites from a trimer of Cre bound to a three-armed (Y-shaped) substrate.
Sau
A K
AK
Section of Molecular Genetics and Microbiology, University of Texas, Austin, Texas 78712, USA.
DeVue Tribble
G
G
Grainge
I
I
Frohlich
R F
RF
Knudsen
B R
BR
Jayaram
M
M
D001483:Base Sequence; D001665:Binding Sites; D002384:Catalysis; D004254:DNA Nucleotidyltransferases / Q000235:genetics / Q000378:metabolism*; D019426:Integrases / Q000378:metabolism*; D007700:Kinetics; D016296:Mutagenesis; D009843:Oligoribonucleotides; D012260:Ribonucleases / Q000378:metabolism*; D014439:Tyramine / Q000378:metabolism; D014443:Tyrosine / Q000378:metabolism*
D009843:Oligoribonucleotides; D014443:Tyrosine; D004254:DNA Nucleotidyltransferases; C045119:FLP recombinase; D019426:Integrases; D012260:Ribonucleases; D014439:Tyramine