Purification and characterization of an initiation protein for chromosomal replication, DnaA, in Bacillus subtilis.
Bacillus subtilis DnaA protein was overproduced by a recombinant plasmid containing B. subtilis dnaA gene in a mutant Escherichia coli strain which is deficient in its own DnaA and RNaseH. The protein was purified to near homogeneity as judged by SDS-PAGE analysis. The purified protein binds preferentially to DNA fragments which are derived from flanking regions of the B. subtilis dnaA gene and contain various numbers of the repeat of 9 nucleotides, TTATCCACA, and closely related sequences. The purified protein binds ATP with high affinity (Kd = 0.02 microM) and ADP with less affinity, but does not bind cAMP. ATP stimulates the binding of the DnaA protein to the repeated sequences. DNaseI footprinting experiments demonstrated that the DnaA bound first to the consensus 9-mer and then to sequences differing by one base from the consensus. Sequences differing by two bases from the consensus were bound by the DnaA only when they were located contiguous to the strong DnaA-boxes. The three DnaA-box clusters, incA, incB, and incC, derived from the replication origin region of the B. subtilis chromosome showed different levels of growth inhibition when they were introduced into B. subtilis. We demonstrated by assaying competition for DnaA-binding among the DnaA-box clusters that there is a good correlation between the degree of growth inhibition by DnaA-box clusters in vivo and their strength of binding to the DNaA protein in vitro.
Fukuoka
T
T
Department of Genetics, Osaka University Medical School.
Moriya
S
S
Yoshikawa
H
H
Ogasawara
N
N
D000255:Adenosine Triphosphate / Q000378:metabolism; D001412:Bacillus subtilis / Q000235:genetics* / Q000378:metabolism; D001426:Bacterial Proteins / Q000235:genetics* / Q000378:metabolism; D001483:Base Sequence; D001667:Binding, Competitive; D002875:Chromosomes / Q000648:ultrastructure*; D004261:DNA Replication; D003850:Deoxyribonuclease I; D005798:Genes, Bacterial; D008969:Molecular Sequence Data; D005810:Multigene Family; D009154:Mutation; D010957:Plasmids; D012091:Repetitive Sequences, Nucleic Acid
D001426:Bacterial Proteins; D000255:Adenosine Triphosphate; D003850:Deoxyribonuclease I