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.
Infection of Escherichia coli dnaB mutants at 41°by phage P1 not only leads to phage production but also to a transient recovery of bacterial DNA replication (1, 2). It was shown that P1 codes for a dnaB analog (ban) protein, which is repressed in P1 wild type and expressed constitutively in Plbac (dnaB analog control) mutants (3). E. coli dnaB(P1 bac) lysogens are able to grow at temperatures that arrest DNA synthesis in the nonlysogen (3). In addition, Plbacban mutants were isolated in which the expression of the viral dnaB analog is prevented. These mutants do not suppress the dnaB character (3). We were interested in the question whether the suppressing action of Plban protein could be imitated by using an in vitro system for DNA replication. Such a system was described for the conversion of 4X174 single-stranded DNA to its duplex form (4). Using E. coli mutants thermosensitive (ts) in DNA synthesis, it was shown that 4X174 DNA duplex formation is dependent on the products of the E. coli genes dnaB, dnaC(D), dnaE, and dnaG. In addition, other proteins are needed for this reaction (5, 6, t).The product of gene dnaB has been purified (4,7,8). In performing initial purification steps (7) using extracts of E. coli dnaB(Plbac) lysogens, we found that an ammonium sulfate fractionated enzyme system contains a sufficient level of replication proteins to replicate OX174 DNA. In such enzyme systems from wild-type or dnaB lysogens the P1 ban protein dominates DNA synthesis, as measured by its temperature resistance.
The constitutive synthesis of the dnaB analog (ban) protein by prophage Plbac permitted the construction of an otherwise non-viable Escherichia coli strain bearing the unsuppressed (sup') amber mutation dnaB266. Growth of this strain is cryosensitive and the ban protein furnished by prophage Plbac does not support bacteriophage I replication [D'Ari et al., J . Mol. Biol. 94,341 -366 (1975)l. Recently a Plbac err prophage mutant was isolated with the ability to confer cryoresistant (err) growth to E. coli sup' dnaB266. The presence of prophage Plbac crr in E. coli supf dnaB266 makes the strain permissive for A growth [D. Touati-Schwartz (1978) Chem. 253, 4746-4753 (1978)l: The ban protein is, however, overproduced by Plbac err when compared to the corresponding Plbac prophage. Overproduction of ban protein results in a thermoresistance of DNA synthesis in crude cell extracts and thermo-resistant heteromultimers composed of ban and thermo-sensitive dnaB subunits. The overproduction of ban protein is also demonstrated in the Plhac err lysogen of E. coli sup+ dnaB266 by precipitation with dnaB antibody.The results suggest that cryosensitive growth of E. coli sup' dnaB266 (Plbac) and its inability to support A replication is due to a cellular deficiency in ban protein molecules rather than to an inefficacy of ban in the absence of an effective dnaB protein.Prophage PI codes for a dnaB analog (ban) protein which is repressed in Escherichia coli containing PI wild type and expressed constitutively in cells containing the P1 dnaB analog control mutant Plbac [1,2]. Plbac ban mutants, on the other hand, fail to synthesize an active ban protein [ l ] . Biochemical studies on the molecular mechanism by which E. coli dnaB mutations are suppressed by prophage Plbac [ l , 21 demonstrated for the first time the presence of ban protein in Plbac lysogens [3 -51. The ban and dnaB subunits were found to be associated with one another in heteromultimers of native molecular weight of approximately 260 000 [3 -51. The ban protein could not be detected in highly purified dnaB preparations from a Plbac ban lysogen [5]. It was concluded that suppression of a thermo-sensitive dnaB mutation by prophage Plbac results from a stabilization of the thermoAbbreviations. ts = thermo-sensitive; i r = thermo-resi~ii~nt ; Mops, 3-(N-morpholino)propane sulfonic acid.sensitive dnaB polypeptide by ban subunits in a heteromultimer [3 -51.The Plbac prophage permitted the construction of an otherwise non-viable strain bearing the unsuppressed amber mutation dnaB266 [l 1. Surprisingly, however, only trace amounts of ban protein were found in such an E. coli supf dnaB266(Plbac) lysogen [5]. In contrast to Plbac lysogens of other E. coli dnaB mutants, this strain has two other unique properties. (a) It is strongly cryosensitive indicating that the ability of ban protein to substitute for dnaB in bacterial growth is limited to temperatures above 30°C [ I ] . (b) It does not permit growth of bacteriophage /z at any temperature suggesting that ban protein...
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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