Uncoupling of the DNA topoisomerase and replication activities of an initiator protein.
The replication initiator proteins encoded by the pT181 and related plasmids have sequence-specific DNA binding and topoisomerase activities. These proteins create a site-specific nick in one strand of the DNA at the origin of replication that serves as a primer for the initiation of replication. To define the regions of the pT181-encoded initiator protein, RepC, that are involved in its DNA binding, topoisomerase, and replication activities, we have carried out site-directed mutagenesis of the repC gene. Analysis of mutant RepC proteins in vitro and in vivo has identified the amino acids that are critical for its various biochemical activities. The DNA binding domain of RepC was found to be located near its C-terminal region and was different from the domain involved in its sequence-specific topoisomerase activity. These studies also showed that the DNA topoisomerase activity of the initiator protein can be uncoupled from its tight noncovalent DNA binding and replication activities.
Dempsey
L A
LA
Department of Molecular Genetics and Biochemistry, University of Pittsburgh School of Medicine, PA 15261.
Birch
P
P
Khan
S A
SA
D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics*; D001483:Base Sequence; D001665:Binding Sites; D004261:DNA Replication; D004264:DNA Topoisomerases, Type I / Q000737:chemistry / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000378:metabolism; D005816:Genetic Complementation Test; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009838:Oligodeoxyribonucleotides / Q000737:chemistry; D010957:Plasmids; D011994:Recombinant Proteins / Q000378:metabolism; D013211:Staphylococcus aureus; D013329:Structure-Activity Relationship
D001426:Bacterial Proteins; D004268:DNA-Binding Proteins; D009838:Oligodeoxyribonucleotides; D011994:Recombinant Proteins; C043650:RepC protein, Staphylococcus aureus; D004264:DNA Topoisomerases, Type I