Abstract
dnaB protein of Escherichia coli is an essential replication protein. A missense mutant has been obtained which results in replacement of an arginine residue with cysteine at position 231 of the protein (P. Shrimankar, L. Shortle, and R. Maurer, unpublished data). This mutant displays a dominant-lethal phenotype in strains that are heterodiploid for dnaB. Biochemical analysis of the altered form of dnaB protein revealed that it was inactive in replication in several purified enzyme systems which involve specific and nonspecific primer formation on single-stranded DNAs, and in replication of plasmids containing the E. coli chromosomal origin. Inactivity in replication appeared to be due to its inability to bind to single-stranded DNA. The altered dnaB protein was inhibitory to the activity of wild type dnaB protein in replication by sequestering dnaC protein which is also required for replication. By contrast, it was not inhibitory to dnaB protein in priming of single-stranded DNA by primase in the absence of single-stranded DNA binding protein. Sequestering of dnaC protein into inactive complexes may relate to the dominant-lethal phenotype of this dnaB mutant.
Highlights
DnaB protein of Escherichia coli is essential for replication of the bacterial chromosome [1,2,3] and of plasmids and bacteriophages such as ColEl [4,5,6], 6x174, and X [7,8,9,10,11] which use this organism as its host
In the identification of genes involved in DNA replication in E. coli, the isolation of conditional mutants defective in DNA synthesis revealed a number of mutants which mapped in the dnaB locus [1].Most of these mutants display a quickstop phenotype consistent with the role of dnaB protein in propagation of the replication fork of the E. coli chromosome
RC231 Is Not Activein D N A Replication-In vitro systems of DNA replication have been described which depend on plasmids containing the E. coli chromosomal origin, oriC [2, 3, 34, 35]
Summary
DnaB protein of Escherichia coli is essential for replication of the bacterial chromosome [1,2,3] and of plasmids and bacteriophages such as ColEl [4,5,6], 6x174, and X [7,8,9,10,11] which use this organism as its host. Proteins required for ABC prepriming in column fractions by immunoblotanalysiswith antisera complex formation (including dnaB protein) were incubated against dnaBprotein (Fig. 2).
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