Mutational analysis of a site-specific recombinase: characterization of the catalytic and dimerization domains of the beta recombinase of pSM19035.
The beta recombinase encoded by the streptococcal plasmid pSM19035, which shows 28 to 34% identity with DNA resolvases and DNA invertases, can catalyze formation of deletions or inversions between properly oriented target sites. We have constructed a number of site-directed mutations at residues that are conserved between the beta protein and other DNA recombinases of the resolvase/invertase family. The analysis of the recombination and DNA-binding ability of each mutant protein shows that the mutations affect the catalytic activity and, in two cases, the dimerization of the protein. The results suggest that the beta protein probably mediates recombination by a catalytic mechanism similar to that proposed for the resolvase/invertase family. Since the beta recombinase differs from DNA resolvases and DNA invertases in its lack of bias towards either of these reactions, the results presented support the hypothesis that its unique properties might depend on details of the architecture or assembly of the recombination complex. In addition, two beta protein mutants that can no longer form dimers in solution have provided new insights into the way the protein binds to DNA.
Canosa
I
I
Centro Nacional de Biotecnologia, C.S.I.C., Campus de la Universidad Autónoma de Madrid, Spain.
Ayora
S
S
Rojo
F
F
Alonso
J C
JC
D000595:Amino Acid Sequence; D001665:Binding Sites / Q000235:genetics; D004254:DNA Nucleotidyltransferases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D004269:DNA, Bacterial / Q000737:chemistry / Q000378:metabolism; D019281:Dimerization; D004926:Escherichia coli / Q000235:genetics; D008969:Molecular Sequence Data; D008970:Molecular Weight; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D009690:Nucleic Acid Conformation; D010957:Plasmids / Q000235:genetics; D011995:Recombination, Genetic; D017386:Sequence Homology, Amino Acid; D013291:Streptococcus / Q000201:enzymology / Q000235:genetics
D004269:DNA, Bacterial; D004254:DNA Nucleotidyltransferases