sbcB Mutations Research Articles

W-reactivation and W-mutagenesis of gamma-irradiated phage lambda

UV irradiation of Escherichia coli wild-type cells manifested the phenomena of W-reactivation (WR) and W-mutagenesis (WM) of phage λ irradiated by 60Co γ-rays in broth. WR of γ-irradiated phage was half as efficient as that of UV-irradiated phage, although the frequency of c mutations in conditions of WR was about the same in both phages. The xthA and recBrecC sbcB mutants were practically identical with wild-type cells in respect of WR and WM of UV- and γ-irradiated phage. As in UV-irradiated phage, WR and WM of γ-irradiated phage were absolutely dependent on the recA + and lexA + genes of the host cell. WR and WM required much smaller doses of UV radiation for induction in polA1 and uvrB mutants. The lig-ts mutant, temperature sensitive in polynucleotide ligase, was deficient in WR and WM of UV- and γ-irradiated phage at the semi-permissive temperature of 37°. The uvrE502 mutant and the allelic recl152 strain were absolutely deficient in WR and WM of γ-irradiated phage. In UV-irradiated phage WR was reduced, but not eliminated, in the uvrE mutant, and WM was entirely suppressed. This is another example of uncoupling of WR and WM which shows that several repair systems are active in WR but only some of them are mutagenic.

The beginning of an investigation of the role of recF in the pathways of metabolism of ultraviolet-irradiated DNA in Escherichia coli.

The recF gene in Escherichia coli was discovered as a locus of mutations which block recombination in a recB − recC − sbcB − strain but not in a recB + recC + sbcB + strain (Horii and Clark, 1974). To understand the significance which was placed on this result it is necessary to know the functions of the recB, recC, and sbcB genes. The recB and recC genes determine exonuclease V (ExoV), an enzyme whose characteristics have been worked out in several laboratories (see e.g. Goldmark and Linn, 1972). Mutations in these genes inactivate ExoV and produce recombination deficiency (see e.g. Clark, 1973) which can be alleviated by sbcB mutations (Kushner et al., 1971). sbcB is thus a gene whose mutations indirectly suppress the recombination deficiency provoked by the absence of ExoV. The product of the sbcB gene is exonuclease I (ExoI) (Kushner et al., 1971, Yajko et al., 1974), an enzyme which degrades single-stranded DNA from 3′-OH termini (Lehman and Nussbaum, 1964). In recB − recC − strains it is presumably the inactivation of ExoI by sbcB mutations which leads to recombination ability, while it is the presence of ExoI which produces recombination deficiency. To explain this, Horii and Clark (1974) hypothesized a pathway of recombination normally inhibited by ExoI which was released from inhibition by the sbcB mutations. This pathway, called the RecF pathway after the recF gene, was considered to be independent of the pathway of recombination involving the recB and recC genes, the RecBC pathway.

The Purification of Exonuclease I from Escherichia coli by Affinity Chromatography

Abstract Escherichia coli exonuclease I has been purified to apparent homogeneity by affinity chromatography. The enzyme has been shown to be exonuclease I by examination of its catalytic and antigenic properties. Its activity is greatly reduced in strains harboring X onA or sbcB mutations. By analysis of acrylamide gels, the enzyme prepared by this method has an apparent molecular weight of 140,000, the protein being a dimer of 70,000 molecular weight subunits.

Transfection by half molecules and inverted molecules of λ DNA: Requirement for exo and β-promoted recombination

A rec + strain of E. coli was infected less efficiently by half molecules and inverted molecules of λ DNA than were recombination deficient strains that lack the recBC DNase. In recB spheroplasts, transfection by half molecules or inverted molecules depends on the action of both of the principal genes of the phage λ system for recombination, namely, the genes for λ exonuclease and β protein. Partial digestion of the ends of inverted molecules by λ exonuclease in vitro bypasses the requirement for the action of either the exo or β gene. Thus, in recB, transfection by broken molecules of DNA with either double-stranded or single-stranded ends does not discriminate between the roles of exonuclease and β protein in genetic recombination. When the recB defect is suppressed by either sbcA or sbcB mutations, neither half molecules nor inverted molecules require λ exonuclease or β protein for infectivity. In recB, the dependence of half molecules and inverted molecules on the genes for exonuclease and β protein for infectivity shows that these genes are active in DNA molecules that are not circular. Furthermore, since the product of the N gene is required for expression of the genes for exonuclease and β protein, circularization of the DNA is probably not a prerequisite for the transcription of N.

Genetic transformation in [formula omitted]: The inhibitory role of the [formula omitted] DNase

Genetic transformation of E. coli for various chromosomal markers was accomplished by (i) using recipient cells that lack the recBC DNase but were recombination proficient due to sbcA or sbcB mutations and (ii) treating the recipient cells with CaCl 2 at a concentration that facilitates transfection by λ DNA. Cotransformation of three markers ( thr + ara +. leu + ) was found to depend on the molecular weight of the transforming DNA.

GENETIC ANALYSIS OF MUTATIONS INDIRECTLY SUPPRESSING recB AND recC MUTATIONS

Mutations in sbcB inactivate exonuclease I and suppress the UV-sensitive, mitomycin-sensitive, recombination-deficient phenotypes associated with recB and recC mutations. Mapping experiments have located sbcB about 0.4 minutes from the his operon at 38.0 on the standard map of E. coli. This places sbcB between supD and his. A four-point cross shows that sbcB lies between P2 attH and his. P2 eduction deleting the his operon beginning with P2 attH also deletes sbcB and produces the expected exonuclease I deficiency and suppression of recB(-). The occurrence of chemical-mutagen-induced and spontaneous mutations indirectly suppressing recB(-) and recC(-) is examined. Three lines of strains produce only sbcA mutations while only sbcB mutations occur in a fourth line. Explanations for this behavior are proposed in light of the ability of the first three lines to express sbcB mutations which they inherit by transduction.