Physiological properties of cold-sensitive suppressor mutations of a temperature-sensitive dnaZ mutant of Escherichia coli

Abstract

Suppressors of a temperature-sensitive dnaZ polymerization mutant of Escherichia coli have been identified by selecting temperature-insensitive revertants. Those suppressed strains which concomitantly became cold sensitive were chosen for further study. Intragenic suppressor mutations, which caused coldsensitive defects in DNA polymerization, were located in dnaZ by transduction with X dnaZ+ phages. Extragenic suppressor mutations were mapped within the initiation gene dnaA. These suppressor-containing strains were defective in initiation at low temperature as determined by measurements of DNA synthesis in vivo and in toluene-treated cells. The occurrence of suppressor mutations of dnaZ(Ts) within the dnaA gene is considered evidence that the dnaA and dnaZ products interact in vivo. A second indication of a dnaA-dnaZ protein-protein interaction was provided by the observation that the introduction of additional copies of the dnaZ+ gene into a strain carrying the dnaA suppressor mutation was lethal [whether the strain was dnaZ+ or dnaZ(Ts)].

Characterization of a thermosensitive mutant of Streptomyces hygroscopicus defective in both DNA and RNA syntheses

“…A heat sensitive defect caused by a single mutation in the uptake or synthesis of a common precursor, needed for both DNA and RNA synthesis (FUCHS et al 1972, FUCHS andNEUHARD 1973) could hardly explain our observation of an immediate stop of both syntheses (Fig. 3A, B, C), because the cellular pool of precursors should allow a limited continuation of syntheses (WECHSLER et al 1973, Born et al 1981, BLINKOVA et al 1983). In addition, in our pulse labelling experiments (Fig.…”

Interactions of DNA replication factors in vivo as detected by introduction of suppressor alleles of dnaA into other temperature-sensitive dna mutants

“…The availability of a strain with TnWO more closely linked to the dnaA(SUZ, Cs) mutation made it possible to demonstrate that the double mutants dnaA(SUZ, Cs) dnaB(Ts) or dnaE(Ts) can be formed and grow, but the frequency of their formation was much lower than when the recipient was dna+ or dnaZ(Ts). These data suggest that dnaA polypeptides interact with each other and with dnaB, -C, -E, and -G. Interaction between dnaA and dnaZ (polymerization protein; also called y [9]) has previously been inferred (2,12,13). We propose that dnaA has at least two functions: one the active function in initiation of rounds of replication and a second function as a component, perhaps structural, of the replication complex in which it associates with many, if not all, of the replication factors.…”

“…These data suggest that the products of the dnaA gene (located near min 82 on the chromosome map) and dnaZ gene (near min 10) interact in vivo. That interpretation is supported by the observation that a dnaA(SUZ, Cs) dnaZ+ strain is killed even at the permissive temperature of 34°C by the introduction of additional copies of the dnaZ+ wild-type gene on a plasmid (2). Interaction between the mutant dnaA(SUZ, Cs) protein and wild-type dnaZ protein could account for this lethality (2).…”

“…Suppressor mutations of a temperature-sensitive (Ts) dnaZ polymerization-defective mutant have been characterized (2,13). These suppressors were isolated by selecting temperatureinsensitive (Ts') revertants and choosing for study only those which concomitantly became cold sensitive (Cs) (i.e., acquired a phenotype in their own right).…”

“…Lethality depends, at least in the case of dnaB(Ts) recipients, on the interaction of three alleles: the incoming dnaA(SUZ, Cs) and the resident dnaA+ and dnaB(Ts). This was proved a Strain ALl was prepared by transducing TnOO at position zib, which is very closely linked to and 95% cotransducible with dnaA, from strain LS1371 [which also carries dnaA46(Ts)] into the dna+ strain GM241 and from there into the dnaA(SUZ, Cs) dnaZ2016(Ts) strain S1 (2), and from there dnaA(SUZ, Cs) and zib::TnlO were transduced into the dna+ strain GM241.…”