DnaA5 protein is thermolabile in initiation of replication from the chromosomal origin of Escherichia coli.
A mutant form of DnaA protein encoded by the dnaA5 allele has been purified and compared biochemically to its wild type counterpart. Biochemical properties of DnaA5 protein include: 1) thermolabile activity in an oriC plasmid replication system dependent on a crude enzyme fraction, 2) comparable affinity relative to DnaA protein in binding to restriction fragments containing either the Escherichia coli chromosomal origin, oriC, or the dnaA promoter, 3) formation of a nucleoprotein complex similar to DnaA protein at the DnaA boxes within the dnaA promoter as detected by protection from DNase I cleavage, 4) formation of an altered nucleoprotein complex with oriC as judged by DNase I protection experiments, 5) inactivity in unwinding of oriC, 6) inactivity in oriC plasmid replication systems dependent on purified enzymes, and 7) inhibition of DnaA protein by addition of DnaA5 protein in assays of replication and of unwinding of oriC, suggesting that mixed complexes formed between wild type and mutant proteins are inactive.
Department of Biochemistry, Michigan State University, East Lansing 48824-1319.
T R
TR
Hupp
J M
JM
Kaguni
D000255:Adenosine Triphosphate / Q000378:metabolism; D001426:Bacterial Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D002876:Chromosomes, Bacterial; D004261:DNA Replication; D004264:DNA Topoisomerases, Type I / Q000378:metabolism; D004269:DNA, Bacterial / Q000096:biosynthesis; D004268:DNA-Binding Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics*; D006358:Hot Temperature; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D016914:Ribonuclease H / Q000378:metabolism
D001426:Bacterial Proteins; D004269:DNA, Bacterial; D004268:DNA-Binding Proteins; C066312:DnaA protein, Bacteria; D000255:Adenosine Triphosphate; D012321:DNA-Directed RNA Polymerases; D016914:Ribonuclease H; D004264:DNA Topoisomerases, Type I