Presence Of RecA Research Articles

Investigation of RecA--polynucleotide interactions from the measurement of LexA repressor cleavage kinetics. Presence of different types of complex.

The proteolysis of the LexA repressor in the presence of RecA and various polynucleotides was studied by measuring the fluorescence decrease of LexA upon cleavage. The results were compared with the DNA binding of RecA to investigate the presence of multiple DNA-RecA complexes. All single-stranded polydeoxyribonucleotides (DNA) efficiently stimulated the proteolysis and the maximum activation was reached in the presence of three or four nucleotides of polynucleotide per monomer of RecA. The stimulative effect was decreased in the presence of larger amounts of poly(dA), poly(dT) or heat-denatured DNA, whereas the excess of single-stranded DNAs chemically modified with chloroacetaldehyde did not present such an inhibitory effect, despite the fact that a second DNA molecule is likely to interact with RecA as monitored by the intrinsic fluorescence of these DNA species. The complicated cleavage promotion and inhibition pattern is tentatively explained by a three-state model assuming that RecA may interact with three single-stranded DNA molecules. According to this model, occupation of the first site would be necessary and sufficient for cleavage promotion, the second site would be neutral with respect to cleavage and the occupation of the third site would inhibit LexA cleavage at least partially. Double-stranded natural DNA did not stimulate cleavage, even under conditions where RecA binds quantitatively to the DNA. No polyribonucleotides (RNA) examined showed a significant stimulative effect either, nor did they appear to interact with RecA.

Survival and mutagenic effects of 5-azacytidine in Escherichia coli

Survival and mutagenesis caused by 5-azacytidine was studied in Escherichia coli. Survival was partially lexA- and recA-dependent and was decreased by the presence of a DNA (cytosine-5)methyltransferase. The dcm, MspI, and EcoRII methyltransferase genes all decreased survival. There was no direct relationship between amount of methylase enzyme present and cell survival, but only plasmids containing a methylase gene sensitized cells to 5-azacytidine. Survival was not affected by uvrA, uvrB or umuCD mutations. Induction of sulA::lacZ fusions by 5-azacytidine was inhibited in strains containing elevated levels of DNA methylase. Cells resistant to 5-azacytidine when they contained a plasmid specifying the EcoRII methylase were sensitive if the plasmid specified the complete EcoRII restriction-modification system. The mechanism of cell death in these situations is therefore different. Mutation of the rpoB gene by 5-azacytidine was studied. The mutation rate was decreased by the presence of recA and lexA mutations. Mutation in umuCD had little effect on the mutation rate. The recA430 mutation, which does not support SOS-dependent mutagenesis induced by UV light, does support 5-azacytidine induced mutagenesis. The presence of DNA (cytosine-5)methyltransferase had no effect on the mutation rate caused by 5-azacytidine treatment. The mutagenic and lethal lesions caused by 5-azacytidine in the absence of methylase therefore differ from the lethal lesions that occur in the presence of methylase. The former could be due to the opening of the 5-azacytosine ring in DNA. Cell death in the presence of methylase could be due to tight binding of methylase to azacytosine containing DNA as well as inhibition of induction of the SOS response.

3' homologous free ends are required for stable joint molecule formation by the RecA and single-stranded binding proteins of Escherichia coli.

The RecA protein of Escherichia coli is important for genetic recombination in vivo and can promote synapsis and strand exchange in vitro. The DNA pairing and strand exchange reactions have been well characterized in reactions with circular single strands and linear duplexes, but little is known about these two processes using substrates more characteristic of those likely to exist in the cell. Single-stranded linear DNAs were prepared by separating strands of duplex molecules or by cleaving single-stranded circles at a unique restriction site created by annealing a short defined oligonucleotide to the circle. Analysis by gel electrophoresis and electron microscopy revealed that, in the presence of RecA and single-stranded binding proteins, a free 3' homologous end is essential for stable joint molecule formation between linear single-stranded and circular duplex DNA.

Deoxyribonucleic acid degradation in vivo and in permeabilized Escherichia coli repair-deficient (recA zab lexA) derivatives.

There are three mutations (recA, zab, and lexA) each of which suppresses the expression of the Escherichia coli tif mutation and causes high deoxyribonucleic acid (DNA) repair deficiency (Castellazi et al., 1972). The effect of the zab mutation on DNA stability was investigated. In vivo, a strain carrying the zab-53 mutation shows (i) no spontaneous DNA degradation and (ii) rapid DNA degradation after ultraviolet irradiation, which depends upon the exonuclease V activity coded by recB+/C+genes and which is independent from the correndonuclease II activity coded by uvrA+/B+. Thus, in regard to DNA stability, the zab mutant behaves like lexA and recA (Howard-Flanders and Boyce, 1966), the latter mutant showing in addition spontaneous DNA breakdown. The degradation patterns of these tif-suppressed strains are shown to be remarkably reproducible in bacteria made permeable to metabolites, by toluene or toluene plus Triton X-100. The degradation properties reflect the activity of the same biochemical system that works in vivo, in that degradation depends upon the presence of recA, zab, or lexA, ultraviolet irradiation, and exonuclease V activity. In addition, adenosine 5'-triphosphate (1 mM) is required. This assay with permeabilized cells offers a useful tool for studying degradation under controlled conditions, especially by permitting the dissociation of energy-dependent from energy-independent steps.