Partial complementation of the UV sensitivity of Deinococcus radiodurans excision repair mutants by the cloned denV gene of bacteriophage T4.

Deinococcus radiodurans has 2 endonucleases that incise UV-irradiated DNA. UV endonuclease-alpha and UV endonuclease-beta, that are believed to functionally overlap. Both endonucleases must be mutationally inactivated to yield an incisionless, markedly UV-sensitive phenotype. denV, the bacteriophage T4 gene encoding pyrimidine dimer-DNA glycosylase (PD-glycosylase), was introduced and expressed via duplication insertion in D. radiodurans wild-type, and single and double UV endonuclease mutants. The strain deficient in UV endonuclease-alpha has wild-type UV resistance, and the expression of PD-glycosylase exerted no survival effect on this strain or wild-type. Expression of denV increased survival of both the markedly UV-sensitive double mutant and the moderately UV-sensitive strain deficient only in UV endonuclease-beta. In endonuclease-beta-deficient cells phenotypic complementation by denV was almost complete in restoring UV resistance to wild-type levels. These results suggest that UV endonuclease-alpha (which is present in the endonuclease-beta-deficient cells) does not recognize one or more types of cyclobutane dimer incised by the PD-glycosylase or UV endonuclease-beta.     

Lysis protein T of bacteriophage T4

Summary Lysis protein T of phage T4 is required to allow the phage's lysozyme to reach the murein layer of the cell envelope and cause lysis. Using fusions of the cloned gene t with that of the Escherichia coli alkaline phosphatase or a fragment of the gene for the outer membrane protein OmpA, it was possible to identify T as an integral protein of the plasma membrane. The protein was present in the membrane as a homooligomer and was active at very low cellular concentrations. Expression of the cloned gene t was lethal without causing gross leakiness of the membrane. The functional equivalent of T in phage is protein S. An amber mutant of gene S can be complemented by gene t, although neither protein R of (the functional equivalent of T4 lysozyme) nor S possess any sequence similarity with their T4 counterparts. The murein-degrading enzymes (including that of phage P22) have in common a relatively small size (molecular masses of ca. 18 000) and a rather basic nature not exhibited by other E. coli cystosolic proteins. The results suggest that T acts as a pore that is specific for this type of enzyme.     

Activities involved in base excision repair of bacteriophage T4 and lambda DNA in vivo

Summary The in vivo excision repair functions of Escherichia coli exonuclease III and 3-methyladenine DNA glycosylase I, and bacteriophage T4 pyrimidine dimer-DNA glycosylase were investigated. Following exposure of bacteriophage T4 or lambda to methyl methanesulfonate or ultraviolet irradiation, survival was determined by plating on E. coli have various genetic backgrounds. Although exonuclease III was shown to participate in base excision repair initiated by 3-methyladenine DNA glcosylase I, it had no detectable role in base excision repair initiated by the T4 pyrimidine dimer-DNA glycosylase. Despite its 3 apurinic/apyrimidinic endonuclease activity in vitro, T4 pyrimidine dimer-DNA glycosylase, even in large quantities, did not complement mutants defective in exonuclease III in the repair of apurinic sites generated by 3-methyladenine DNA glycosylase I in vivo.     

Regulation of the yeast RAD2 gene DNA damage-dependent induction correlates with protein binding to regulatory sequences and their deletion influences survival

Summary In the yeast Saccharomyces cerevisiae the RAD2 gene is absolutely required for damage-specific incision of DNA during nucleotide excision repair and is inducible by DNA-damaging agents. In the present study we correlated sensitivity to killing by DNA-damaging agents with the deletion of previously defined specific promoter elements. Deletion of the element DRE2 increased the UV sensitivity of cells in both the G₁/early S and S/G₂ phases of the cell cycle as well as in stationary phase. On the other hand, increased UV sensitivity associated with deletion of the sequence-related element DRE1 was restricted to cells irradiated in G₁/S. Specific binding of protein(s) to the promoter elements DRE1 and DRE2 was observed under non-inducing conditions using gel retardation assays. Exposure of cells to DNA-damaging agents resulted in increased protein binding that was dependent on de novo protein synthesis.     

The Escherichia coli recA gene increases resistance of the yeast Saccharomyces cerevisiae to ionizing and ultraviolet radiation

Summary The Escherichia coli recA protein coding region was ligated into an extrachromosomally replicating yeast expression vector downstream of the yeast alcohol dehydrogenase promoter region to produce plasmid pADHrecA. Transformation of the wild-type yeast strains YNN-27 and 7799-4B, as well as the recombination-deficient rad52-t C5-6 mutant, with this shuttle plasmid resulted in the expression of the bacterial 38 kDa RecA protein in exponential phase cells. The wild-type YNN27 and 7799-4B transformants expressing the bacterial recA gene showed increased resistance to the toxic effects of both ionizing and ultraviolet radiation. RecA moderately stimulated the UV-induced mutagenic response of 7799-4B cells. Transformation of the rad52-t mutant with plasmid pADHrecA did not result in the complementation of sensitivity to ionizing radiation. Thus, the RecA protein endows the yeast cells with additional activities, which were shown to be error-prone and dependent on the RAD52 gene.     

Influence of DNA repair defects (rad1, rad52) on nitrogen mustard mutagenesis in yeast.

Summary Nitrogen mustard (HN2) mutagenesis of a plasmid-borne copy of the Saccharomyces cerevisiae SUP4-o gene was examined in a repair-proficient yeast strain and isogenic derivatives defective for excision (radl) or DNA double-strand break (rad52) repair. The excision repair deficiency sensitized the cells to killing by HN2 and abolished mutation induction. Inactivation of RAD52 had no influence on the lethality of HN2 treatment but diminished the induced mutation frequency by 50% at all doses tested. DNA sequence analysis of HN2-induced SUP4-o mutations suggested that RAD52 contributed to the production of basepair substitutions at G·C sites. The rad52 defect appeared to alter the distribution of G·C A·T transitions in SUP4-o relative to the distribution for the wild-type strain. This difference did not seem to be due to an effect of RAD52 on the relative fractions of HN2-induced transitions at localized (flanked by A·T pairs) or contiguous (flanked by at least one G·C pair) G·C sites but instead to an influence on the strand specificity of HN2 mutagenesis. In the repair-proficient strain, the transitions showed a small bias for sites having the guanine on the transcribed strand and this preference was eliminated by inactivation of RAD52.     

Recombinant expression of maize nucleotide excision repair protein Rad23 in Escherichia coli

Nucleotide excision is a highly conserved DNA repair pathway for correcting DNA lesions that cause distortion of the double helical structure. The protein heterodimer XPC-Rad23 is involved in recognition of and binding to such lesions. We have isolated full-length cDNAs encoding two different members of the maize Rad23 family. The deduced amino acid sequences of both maize orthologues show a high degree of homology to plant and animal Rad23 proteins. The cDNA encoding maize Rad23A was cloned as an in-frame C-terminal fusion of glutathione S-transferase. This chimera was expressed in Escherichia coli as a soluble protein and purified to homogeneity using glutathione-agarose followed by MonoQ column chromatography. Purified recombinant maize Rad23 protein was used to generate polyclonal antibodies that cross-react with a approximately 48-kDa protein in extracts from plant as well as mammalian cells. The purified recombinant protein and antibodies would be useful reagents to study the biochemistry of nucleotide excision repair in plants.     

Efficient UV stimulation of yeast integrative transformation requires damage on both plasmid strands

The nature of UV-induced pre-recombinational structures was studied using transformation of Saccharomyces cerevisiae cells with non-replicative plasmids. Transformation by double-stranded plasmids irradiated with UV was stimulated up to 50-fold, and both plasmid integration and conversion of the mutated chromosomal selective gene were found to be equally increased. The stimulation observed with such totally irradiated plasmids was not found with plasmids bearing lesions in only one strand. This effect is attributed to the formation by excision repair of recombinogenic structures consisting of a pyrimidine dimer opposite a gap. When single-stranded integrative plasmids were irradiated, their transforming potential was decreased but the proportion of transformants that arose by gene conversion, rather than by plasmid integration, was increased from 8% to 49% as a function of the UV dose. Possible reasons why single-strand UV lesions favour gene conversion are discussed.     

The PSO4 gene is responsible for an error-prone recombinational DNA repair pathway in Saccharomyces cerevisiae

Summary The induction of mitotic gene conversion and crossing-over inSaccharomyces cerevisiae diploid cells homozygous for thepso4-1 mutation was examined in comparison to the corresponding wild-type strain. Thepso4-1 mutant strain was found to be completely blocked in mitotic recombination induced by photoaddition of mono- and bifunctional psoralen derivatives as well as by mono- (HN1) and bifunctional (HN2) nitrogen mustards or 254 nm UV radiation in both stationary and exponential phases of growth. Concerning the lethal effect, diploids homozygous for thepso4-1 mutation are more sensitive to all agents tested in any growth phase. However, this effect is more pronounced in the G2 phase of the cell cycle. These results imply that the ploidy effect and the resistance of budding cells are under the control of thePSO4 gene. On the other hand, thepso4-1 mutant is mutationally defective for all agents used. Therefore, thepso4-1 mutant has a generalized block in both recombination and mutation ability. This indicates that thePSO4 gene is involved in an error-prone repair pathway which relies on a recombinational mechanism, strongly suggesting an analogy between thepso4-1 mutation and theRecA orLexA mutation ofEscherichia coli.     

Accumulation and secretion of exoglucanase activity in yeast secretory mutants

Representative conditional yeast secretory mutants, blocked in transport of secretory and plasma membrane proteins from the endoplasmic reticulum (sec 18), from the Golgi body (sec 7) and in transport of secretory vesicles (sec 1), accumulated exoglucanase, a constitutive yeast activity, when incubated at the restrictive temperature (37°C). Different proportions of the accumulated activity were released by mutant cells under permissive conditions. The presence or absence of cycloheximide during the secretion period made no differences in the results. More than 90% of the internal activity was bound to membrane in wild type cells. However, only the soluble pool underwent changes during the accumulation or secretion periods. The bulk of secretory invertase accumulated by sec 1 was also soluble. By contrast sec 7 and sec 18 accumulated membrane-bound as well as soluble invertase forms and both were secreted in similar proportions in each mutant. More than 90% of the accumulated invertase was secreted at the permissive temperature in sec 18 cells. That percentage was significantly lower for exoglucanase (<65%). Concomitantly, invertase accumulated by this mutant exited from the cells with a lower half time (t 1/2=150 min). These results may be interpreted assuming that exoglucanase is exported by a passive flow of the soluble pool.Non-standard abbreviations p-NPG p-nitrophenyl--d-glucopyranoside - Con A concanavalin A - Tris tris(hydroxymethyl)-amino-methane     

Chlorella virus PBCV-1 encodes a homolog of the bacteriophage T4 UV damage repair gene denV.

The bacteriophage T4 denV gene encodes a well-characterized DNA repair enzyme involved in pyrimidine photodimer excision. We have discovered the first homologs of the denV gene in chlorella viruses, which are common in fresh water. This gene functions in vivo and also when cloned in Escherichia coli. Photodamaged virus DNA can also be photoreactivated by the host chlorella. Since the chlorella viruses are continually exposed to solar radiation in their native environments, two separate DNA repair systems, one that functions in the dark and one that functions in the light, significantly enhance their survival.     

The bacteriophage T4 dexA gene: sequence and analysis of a gene conditionally required for DNA replication

Summary We have cloned and sequenced a bacteriophage T4 EcoRI fragment that complements T4 del (39-56) infections of an optA defective Escherichia coli strain. Bacteria containing this recombinant plasmid synthesize two new proteins with molecular weights of 9 and 26 kilodaltons. We have identified the gene encoding the 26 kilodalton protein as essential for T4 infections of optA defective E. coli. Genetic and biochemical results are consistent with the identification of this protein as the product of the dexA gene, which encodes a 3 to 5 exonuclease.     

Isolation and genetic characterization of new uvsW alleles of bacteriophage T4

Summary TheuvsW gene of bacteriophage T4 is required for wild-type levels of recombination, for normal survival and mutagenesis after UV irradiation, and for wild-type resistance to hydroxyurea. Additionally,uvsW mutations restore the arrested DNA synthesis caused by mutations in any of several genes that block secondary initiation (recombination-primed replication, the major mode of initiation at late times), but only partially restore the reduced burst size. AuvsW deletion mutation was constructed to establish the null-allele phenotype, which is similar but not identical to the phenotype of the canonicaluvsW mutation, and to demonstrate convincingly that theuvsW gene is non-essential (althoughuvsW mutations severely compromise phage production). In an attempt to uncouple the diverse effects ofuvsW mutations, temperature-sensitiveuvsWts mutants were isolated. Recombination and replication effects were partially uncoupled in these mutants, suggesting distinct and separable roles foruvsW in the two processes. Furthermore, the restoration of DNA synthesis but not recombination in the double mutantsuvsW uvsX anduvsW uvsY prompts the hypothesis that the restored DNA synthesis is not recombinationally initiated.