Abstract
We have determined the nucleotide sequence of the dnaB gene and the primary structure of the dnaB protein of Escherichia coli (Arai, K., Yasuda, S., and Kornberg, A. (1981) J. Biol. Chem. 256, 5247-5252). The coding region for the dnaB protein is 1413 base pairs followed by double stop codons and preceded by a possible promoter sequence. The dnaB gene lacks a typical Shine-Dalgarno sequence. The primary structure deduced from the DNA sequence is consistent with the protein chemical data. The dnaB protein contains 470 amino acid residues and has a calculated molecular weight of 52,265. In the mature protein, the initiator methionine residue is removed in vivo leaving alanine as the NH2-terminal residue. Based on the amino acid sequence, we predict that the dnaB protein may be composed of two domains. A hydrophilic NH2-terminal region (residues 1-20) is followed by a compact domain and a possible hinge region (residues 21-172) consisting primarily of alpha-helix. The sites of facile tryptic cleavage are at the arginine residues at 14 and 171. The DNA-dependent ATPase domain (residues 172-470) is located at the COOH-terminal end of the protein.
Highlights
We have determined the nucleotide sequence of the DNA binding, and interaction with dnaC protein, primase, dnaB gene and the primary structure of the dnaB and other prepriming proteins
The coding region for the dnaB protein is 1413 base pairs followed by double stop codons and preceded by a possible promoter sequence
A polypeptidefragment of M, = 33,000, from theCOOH-terminalportion of the protein, elicits DNA-dependent ATPase activity. These results suggest that the dnaB protein is composed of at least two functional domains
Summary
Bacterial Strains and Plasmids-E. coli YSlrecA (thr, leu, minA, s t r , thi, endA, recA56, srl) (TnlO) harboring the high copy number plasmidpKAl(5) was used for thepreparation of dnaB protein. The dnaB protein is a hexamer (4,5 ) with a monomer molecular weight of 50,000 ( 5 ) .Based on its role in phage 4x174 replication, theproteinservesintheprimingstage of discontinuous replication and prepares the DNA for primer synthesis with other components in the primosome (6-10). In addition to promoting theconversion of the 4x174 single-stranded to the circular double-stranded replicative form, the dnaB protein exhibits multiple activitiesin vitro, namely: ( a )DNA-dependent ATPase(11,12);( b )ATP binding (13);( c )single-stranded DNA binding(13); ( d ) interactions with dnaC protein (14, 15), phage X-encoded P protein (16), and phage P1-encoded ban protein (17); and ( e ) general priming with primase (9). Multiple functions for the dnaB protein have been suggested in i n vivo studies. An altered dnaB protein derived from aninitiationmutant, dnaB252 (3), retains DNA&-
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
