J. Biochem, 1990, Vol. 107, No. 5 732-739
© 1990 Japanese Biochemical Society
research-article |
Purification and Characterization of an Initiation Protein for Chromosomal Replication, DnaA, in Bacillus subtilis1
Department of Genetics, Osaka University Medical School Kita-ku, Osaka, Osaka 530
2 To whom correspondence should be addressed.
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 amnity (Kd, =0.02 µM) and ADP with less affinity, but does not bind cAMP. ATP stimulates the binding of the DnaA protein to the repeated sequences. DNasel 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.
1 This work was supported by Grants-in-Aid for Scientific Research on Priority Areas and for Scientific Research from the Ministry of Education, Science and Culture of Japan.
CiteULike 
Connotea 
Del.icio.us What's this?
This article has been cited by other articles:
|
|
 |
|
  S. Ishikawa, Y. Ogura, M. Yoshimura, H. Okumura, E. Cho, Y. Kawai, K. Kurokawa, T. Oshima, and N. Ogasawara Distribution of Stable DnaA-Binding Sites on the Bacillus Subtilis Genome Detected using a Modified ChIP-chip Method DNA Res, October 10, 2007; (2007) dsm017v1. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 N. Ichihashi, K. Kurokawa, M. Matsuo, C. Kaito, and K. Sekimizu Inhibitory Effects of Basic or Neutral Phospholipid on Acidic Phospholipid-mediated Dissociation of Adenine Nucleotide Bound to DnaA Protein, the Initiator of Chromosomal DNA Replication J. Biol. Chem., August 1, 2003; 278(31): 28778 - 28786. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 Y. Ogura, Y. Imai, N. Ogasawara, and S. Moriya Autoregulation of the dnaA-dnaN Operon and Effects of DnaA Protein Levels on Replication Initiation in Bacillus subtilis J. Bacteriol., July 1, 2001; 183(13): 3833 - 3841. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D. Ishigo-oka, N. Ogasawara, and S. Moriya DnaD Protein of Bacillus subtilis Interacts with DnaA, the Initiator Protein of Replication J. Bacteriol., March 15, 2001; 183(6): 2148 - 2150. [Abstract] [Full Text]
|
|
|
|
|
|
K. S. Doran, D. R. Helinski, and I. Konieczny A Critical DnaA Box Directs the Cooperative Binding of the Escherichia coli DnaA Protein to the Plasmid RK2 Replication Origin J. Biol. Chem., June 18, 1999; 274(25): 17918 - 17923. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
R. Caspi, D. R. Helinski, M. Pacek, and I. Konieczny Interactions of DnaA Proteins from Distantly Related Bacteria with the Replication Origin of the Broad Host Range Plasmid RK2 J. Biol. Chem., June 9, 2000; 275(24): 18454 - 18461. [Abstract] [Full Text] [PDF]
|
|