NOVEL Escherichia coli dnaB mutant: direct involvement of the dnaB252 gene product in the synthesis of an origin-ribonucleic acid species during initiaion of a round of deoxyribonucleic acid replication

Abstract

The initiation process of deoxyribonucleic acid (DNA) replication in Escherichia coli has been studied using the thermoreversible dna initiation mutant E. coli HfrH165/120/6 dna-252. This dna mutation was incorrectly classed as a dnaA mutation. Biochemical and genetic evidence suggests that the dna-252 mutant is a novel dnaB mutant, possessing phenotypic properties which distinguish it from other dnaB mutants. Sensitivity of reinitiation in the dna-252 mutant to specific inhibitors of protein, ribonucleic acid (RNA), and DNA synthesis was studied. Reinitiation is shown to be sensitive to rifampin and streptolydigin but not to chloramphenicol. Thus, the dna-252 gene product appears to be required during the initiation process for a step occurring either before or during synthesis of an RNA species (origin-RNA). Using reversible inhibition of RNA synthesis by streptolydigin of a streptolydigin-sensitive derivative of the dna-252 mutant, the dna-252 gene product is shown to be directly involved in the synthesis of an origin-RNA species. These results are included in a schematic model presented in the accompanying paper of the temporal sequence of events occurring during the initiation process. 1476 on August 1, 2020 by guest

Chromosome structure of Escherichia coli mutants temperature sensitive for deoxyribonucleic acid replication

“…The dnaB252 mutant bacteria accumulated a structure sedimenting at 1,420S. These data confirm the conclusion of Zyskind and Smith (17) which states that the dnaB252 allele affects initiation of replication.…”

“…A reversible dna mutant carrying a lesion in the dnaB gene, which affects initiation of replication (17), was examined to determine if its chromosome conformation resembled that of the dnaA mutant. Sedimentation profiles ofthe folded chromosomes from the dnaB strain, HfrH165/120/6, after incubation at 30 and 430C for 90 min are shown in Fig.…”

dnaA protein-regulated transcription: effects on the in vitro replication of Escherichia coli minichromosomes.

“…This transcription is affected by dnaA protein. From genetical and physiological data it has been inferred that dnaA protein acts at an early stage of initiation (Zyskind et al, 1977;Kung and Glaser, 1978). Second site mutations are found in rpoB (Bagdasarian et al, 1977;Atlung, 1981).…”

Deoxyribonucleic acid initiation mutation dnaB252 is suppressed by elevated dnaC+ gene dosage

“…The dna-252 mutation renders Escherichia coli cells defective in the initiation of DNA replication. Clfication of this mutation as a dnaB allele is based on the following evidence: (i) the map position is in the dnaB region of the chromosome (20); (ii) extracts of the mutant can be complemented by dnaB protein in a OX174 in vitro assay system (S. Wickner,cited in 20,5); (iii) extracts of the mutant lack dnaB-complementing activity (5); and (iv) the phenotype of the mutant is suppressed in P1 lysogens which overproduce the phage ban protein, an analog of dnaB (5). The dnaB protein encoded by dnaB252 is the only mutant dnaB protein which retains the associated ATPase activity (5).…”