Mutagenized E. coli B/r cells were subjected to a procedure designed to select mutants temperature-sensitive for initiation of deoxyribonucleic acid (DNA) replication. Seventeen mutants exhibiting limited residual DNA synthesis at 42 C were obtained and the dnasites were mapped genetically. Sixteen of the sites map near dnaA, dnaB, and dnaC. One mutant (dna-208) maps in a new location between the trp and his genes. We propose to call this mutant dnaI208. In complementation experiments dnaC+ and dnaI+ were dominant to dnaCand dnaI alleles, respectively. However, dnaAwas dominant to the wild-type allele dnaA+. All dnaA mutants and four out of six dnaC mutants could be suppressed by F factor integration. The pattern of suppression was specific for each mutant.
An Escherichia coli K12 dnaB dnaC mutant was constructed by P1 transduction of the dnaC allele into a dnaB recipient stain. dnaB dnaC transductant were discriminated from dnaB mutants by their inability to grow at 40 degree C after lysogenization with phage P1bac. The dnaB dnaC mutant character was verified by 1. P1 transduction, and 2. by in vitro complementation with dnaB and dnaC wild type protein fractions. DNA synthesis was studied in strains containing dnaB, dnaB dnaC alleles in an otherwise uniform genetic background with the dnaB character either unsuppressed or suppressed by P1bac prophage. Degradation at 42 degree C of [3H]-thymidine pulse-labeled DNA in dnaB and dnaB dnaC mutants is suppressed by P1bac. However, unlike the dnaC mutant, the P1bac lysogen of the dnaB dnaC mutant exhibits an abrupt cessation of DNA synthesis and less residual cell divisions at 42 degree C indicating an inhibition of DNA chain elongation rather than a defect in DNA initiation. It is suggested that denaturation of the dnaB protein effects the dnaC function.
High-temperature treatment of thermosensitive
dna
mutants lysogenic for phage λ leads to prophage induction and release of phage (at the permissive temperature) in elongation-defective mutants of the genotypes
dnaB, dnaE
, and
dnaG.
In initiation-defective mutants no prophage induction occurs at 42 C in mutants of the genotype
dnaA
, whereas with a
dnaC
mutant as well as with strain HfrH 252 (map position not yet known) phages are released at 42 C. DNA degradation at the replication fork at 42 C is observed in all
dnaB
(λ) mutants tested, but not in mutants of the genotypes
dnaE
(λ) and
dnaG
(λ). Therefore, degradation of replication fork DNA is not a prerequisite for prophage induction.
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