Molecular cloning and analysis of Schizosaccharomyces pombe rad9, a gene involved in DNA repair and mutagenesis

Summary The mutant allele rad9-192 renders Schizosaccharomyces pombe cells sensitive to ionizing radiation and UV light. We have isolated from a S. pombe genomic DNA library a unique recombinant plasmid that is capable of restoring wild-type levels of radioresistance to a rad9 192-containing cell population. Plasmid integration studies using the cloned DNA, coupled with mating and tetrad analyses, indicate that this isolated DNA contains the wild-type rad9 gene. We inactivated the repair function of the cloned fragment by a single insertion of the S. pombe ura4 gene. This nonfunctional fragment was used to create a viable disruption mutant, thus demonstrating that the rad9 gene does not encode an essential cellular function. In addition, the rad9-192 mutant population is as radiosensitive as the disruption mutant, indicating that rad9 gene function is severely if not totally inhibited by the molecular defect responsible for the rad9-192 phenotype. DNA sequence analysis of rad9 reveals an open reading frame of 1,278 bp, interrupted by three introns 53 bp, 57 bp, and 56 by long, respectively, and ending in the termination codon TAG. This gene is capable of encoding a protein of 426 amino acids, with a corresponding calculated molecular weight of 47,464 daltons. No significant homology was detected between the rad9 gene or its deduced protein sequence and sequences previously entered into DNA and protein sequence data banks.     

Construction of recA mutants of Acholeplasma laidlawii by insertional inactivation with a homologous DNA fragment

Mycoplasmas (class Mollicutes) are wall-less prokaryotes phylogenetically related to gram-positive bacteria. This study describes the construction of recA mutants of the mycoplasma Acholeplasma laidlawii. An internal fragment of the recA gene from A. laidlawii was cloned into a plasmid that does not replicate in this organism. When this plasmid construct was used to transform A. laidlawii, it inserted into the chromosome, disrupting the recA gene. The pheno-type of the resulting recA mutant was compared to that of wild-type cells and to that of a strain that has a naturally occurring ochre mutation in its recA gene. As found in other bacterial systems, loss of RecA activity resulted in cells deficient in DNA repair.     

Cloning of theHelicobacter pylori recA gene and functional characterization of its product

The RecA protein is a key enzyme involved in DNA recombination in bacteria. Using a polymerase chain reaction (PCR) amplification we cloned arecA homolog fromHelicobacter pylori. The gene revealed an open reading frame (ORF) encoding a putative protein of 37.6 kDa showing closest homology to theCampylobacter jejuni RecA (75.5% identity). A putative ribosome binding site and a near-consensus σ⁷⁰ promoter sequence was found upstream ofrec A. A second ORF, encoding a putative protein with N-terminal sequence homology to prokaryotic and eukaryotic enolases, is located directly downstream ofrecA. Compared to the wild-type strains, isogenicH. pylori recA deletion mutants of strains 69A and NCTC11637 displayed increased sensitivity to ultraviolet light and abolished general homologous recombination. The recombinantH. pylori RecA protein produced inEscherichia coli strain GC6 (recA ⁻) was 38 kDa in size but inactive in DNA repair, whereas the corresponding protein inH. pylori 69A migrated at the greater apparent molecular weight of approx. 40 kDa in SDS-polyacrylamide gels. However, complementation of theH. pylori mutant using the clonedrecA gene on a shuttle vector resulted in a RecA protein of the original size and fully restored the general functions of the enzyme. These data can be best explained by a modification of RecA inH. pylori which is crucial for its function. The potential modification seems not to occur when the protein is produced inE. coli, giving rise to a smaller but inactive protein.     

Partial complementation of the UV sensitivity of E. coli and yeast excision repair mutants by the cloned denV gene of bacteriophage T4

Summary The denV gene of bacteriophage T4 was reconstituted from two overlapping DNA fragments cloned in M13 vectors. The coding region of the intact gene was tailored into a series of plasmid vectors containing different promoters suitable for expression of the gene in E. coli and in yeast. Induction of the TAC promoter with IPTG resulted in overexpression of the gene, which was lethal to E. coli. Expression of the TACdenV gene in the absence of IPTG, or the use of the yeast GAL1 or ADH promoters resulted in partial complementation of the UV sensitivity of uvrA, uvrB, uvrC and recA mutants of E. coli and rad1, rad2, rad3, rad4 and rad10 mutants of S. cerevisiae. The extent of denV-mediated reactivation of excision-defective mutants was approximately equal to that of photoreactivation of such strains. Excision proficient E. coli cells transformed with a plasmid containing the denV gene were slightly more resistant to ultraviolet (UV) radiation than control cells without the denV gene. On the other hand, excision proficient yeast cells were slightly more sensitive to killing by UV radiation following transformation with a plasmid containing the denV gene. This effect was more pronounced in yeast mutants of the RAD52 epistasis group.     

Use of recA803, a partial suppressor of recF, to analyze the effects of the mutant Ssb (single-stranded DNA-binding) proteins in vivo and in vitro

Summary We examined the possibility that the ssb-1 and ssb-113 mutants exert some of their effects by interfering with the normal function of wild-type RecF protein. Consistent with this possibility, we found that recA803, which partially suppresses recF mutations, also partially suppresses both ssb mutations, as detected by an increase in UV resistance. No evidence was obtained for suppression of the defect in lexA regulon inducibility caused by the ssb mutations. Consequently we suggest that suppression occurs by increasing recombinational repair. In vitro tests of Ssb mutant and wild-type proteins revealed that the single-stranded DNA dependent ATPase activity of RecA protein is more susceptible to inhibition than the joint-molecule-forming activity. All three Ssb proteins inhibit the ATPase activity of RecA wild-type protein almost completely while under similar conditions they inhibit the joint-molecule-forming activity only slightly. Both activities of RecA803 protein were found to be less inhibited by the three Ssb proteins than those of RecA wild-type protein. This is consistent with the suppressing ability of recA803. We found no evidence to contradict the previously proposed hypothesis that ssb-1 affects recombinational repair by acting as a weaker form of Ssb protein. We found, however, only very weak evidence that Ssb-113 protein interferes directly with recombinational repair so that the possibility that it interferes with a normal function of RecF protein must remain open.     

Cloning and characterization of the Azotobacter vinelandii recA gene and construction of a recA deletion mutant

Summary The recA gene of Azotobacter vinelandii was isolated from a genomic library by heterologous complementation of an Escherichia coli recA mutation for resistance to UV radiation. The A. vinelandii recA gene was localized on adjacent PstI fragments of 1.3 and 1.7 kb. The cloned A. vinelandii recA gene was functionally analogous to the E. coli recA gene. It was also able to complement the E. coli recA mutation for homologous recombination. A recA deletion mutant of A. vinelandii was constructed. This mutant was sensitive to DNA-damaging agents like UV rays, methyl methane sulfonate (MMS) and nalidixic acid and was deficient in homologous recombination.     

Molecular cloning, sequence and regulation of expression of the recA gene of the phototrophic bacterium Rhodobacter sphaeroides

The recA gene of Rhodobacter sphaeroides 2.4.1 has been isolated by complementation of a UV-sensitive RecA^– mutant of Pseudomonas aeruginosa. Its complete nucleotide sequence consists of 1032 bp, encoding a polypeptide of 343 amino acids. The deduced amino acid sequence displayed highest identity to the RecA proteins from Rhizobium mehloti, Rhizobium phaseoli, and Agrobacterium tumefaciens. An Escherichia coli-like SOS consensus region, which functions as a binding site for the LexA repressor molecule was not present in the 215 by upstream region of the R. sphaeroides recA gene. Nevertheless, by using a recA-lacZ fusion, we have shown that expression of the recA gene of R. sphaeroides is inducible by DNA damage. A recA-defective strain of R. sphaeroides was obtained by replacement of the active recA gene by a gene copy inactived in vitro. The resulting recA mutant exhibited increased sensitivity to UV irradiation, and was impaired in its ability to perform homologous recombination as well as to trigger DNA damage-mediated expression. This is the first recA gene from a Gram-negative bacterium that lacks an E. coli-like SOS box but whose expression has been shown to be DNA damage-inducible and auto-regulated.     

Operator-constitutive mutation in the <Emphasis Type="Italic">recA</Emphasis> gene enhances radiation resistance of <Emphasis Type="Italic">Escherichia coli</Emphasis>

Plasmid pUC19-recAo^c carrying a mutant allele of the recA gene, which plays the key role in the control of the SOS repair system and homologous recombinational repair, causes a 1.5-fold increase in radiation resistance of Escherichia coli ΔrecA cells, as compared to the wild-type recA ⁺ cells. The protective effect of this plasmid is drastically reduced in mutant lexA3 recAΔ21 deficient in the LexA protein and in induction of the SOS regulon. Plasmid pUC19-recAo^c effectively suppresses UV sensitivity of the ΔrecA mutant. Mutation recAo20 allows constitutive high-level synthesis of the RecA protein. This mutation impairs the SOS box in the operator site of the recA gene and enhances heterology of the dimer LexA binding site. These data confirm that high level of the RecA protein synthesis per se is not sufficient for the expression of γ-inducible functions and that the derepression of lexA-dependent genes, other than recA gene, is necessary for the complete induction of the SOS repair system.     

Analysis of deletion mutations of the rpsL gene in the yeast Saccharomyces cerevisiae detected after long-term flight on the Russian space station Mir

Using the yeast Saccharomyces cerevisiae on board the Russian space station Mir, we studied the effects of long-term space flight on mutation of the bacterial ribosomal protein L gene (rpsL) cloned in a yeast-Escherichia coli shuttle vector. The mutation frequencies of the cloned rpsL gene on the Mir and the ground (control) yeast samples were estimated by transformation of E. coli with the plasmid DNAs recovered from yeast and by assessment of the conversion of the rpsL wild-type phenotype (Sm^S) to its mutant phenotype (Sm^R). After a 40-day space flight, some part of space samples gave mutation frequencies two to three times higher than those of the ground samples. Nucleotide sequence analysis showed no apparent difference in point mutation rates between the space and the ground mutant samples. However, the greater part of the Mir mutant samples were found to have a total or large deletion in the rpsL sequence, suggesting that space radiation containing high-linear energy transfer (LET) might have caused deletion-type mutations.     

The repair of double-strand breaks in the nuclear DNA of Saccharomyces cerevisiae and its genetic control

Summary With the use of neutral sucrose sedimentation techniques, the size of unirradiated nuclear DNA and the repair of double-strand breaks induced in it by ionizing radiation have been determined in both wild-type and homozygous rad52 diploids of the yeast Saccharomyces cerevisiae. The number average molecular weight of unirradiated DNA in these experiments is 3.0×10⁸±0.3 Daltons. Double-strand breaks are induced with a frequency of 0.58×10^-10 per Daltonkrad in the range of 25 to 100 krad. Since repair at low doses is observed in wild-type but not homozygous rad52 strains, the corresponding rad52 gene product is concluded to have a role in the repair process. Cycloheximide was also observed to inhibit repair to a limited extent indicating a requirement for protein synthesis. Based on the sensitivity of various mutants and the induction frequency of double-strand breaks, it is concluded that there are 1 to 2 double-strand breaks per lethal event in diploid cells incapable of repairing these breaks.     

Mutational analysis of the Streptomyces lividans recA gene suggests that only mutants with residual activity remain viable

Temperature-sensitive integration plasmids carrying internal fragments of the Streptomyces lividans TK24 recA gene were constructed and used to inactivate the chromosomal recA gene of S. lividans by gene disruption and gene replacement. Integration of these plasmids resulted in recA mutants expressing C-terminally truncated RecA proteins, as deduced from Southern hybridization experiments. Mutants FRECD2 in which the last 42 amino acids, comprising the variable part of bacterial RecA proteins, had been deleted retained the wild-type phenotype. The S. lividans recA mutant FRECD3 produced a RecA protein lacking 87 amino acids probably including the interfilament contact site. FRECD3 was more sensitive to UV and MMS than the wild-type. Its ability to undergo homologous recombination was impaired, but not completely abolished. Integration of the disruption plasmid pFRECD3 in S. coelicolor“Müller” caused the same mutant phenotype as S. lividans FRECD3. In spite of many attempts no S. lividans recA mutants with deletions of 165 C-terminal amino acids or more were isolated. Furthermore, the recA gene could not be replaced by a kanamycin resistance cassette. These experiments indicate a crucial role of the recA gene in ensuring viability of Streptomyces. Received: 20 December 1996 / Accepted: 25 March 1997     

Characterization of recA genes and recA mutants of Rhizobium meliloti and Rhizobium leguminosarum biovar viciae

Summary DNA fragments carrying the recA genes of Rhizobium meliloti and Rhizobium leguminosarum biovar viciae were isolated by complementing a UV-sensitive recA ^– Escherichia coli strain. Sequence analysis revealed that the coding region of the R. meliloti recA gene consists of 1044 by coding for 348 amino acids whereas the coding region of the R. leguminosarum bv. viciae recA gene has 1053 bp specifying 351 amino acids. The R. meliloti and R. leguminosarum bv. viciae recA genes show 84.8% homology at the DNA sequence level and of 90.1% at the amino acid sequence level. recA ^– mutant strains of both Rhizobium species were constructed by inserting a gentamicin resistance cassette into the respective recA gene. The resulting recA mutants exhibited an increased sensitivity to UV irradiation, were impaired in their ability to perform homologous recombination and showed a slightly reduced growth rate when compared with the respective wild-type strains. The Rhizobium recA strains did not have altered symbiotic nitrogen fixation capacity. Therefore, they represent ideal candidates for release experiments with impaired strains.The accession numbers: X59956 R. LEGUMINOSARUM REC A ALAS-DNA; X59957 R. MELITOTI REC A ALAS-DNA     

Cloning and characterisation of the recA gene of Agrobacterium tumefaciens C58

Summary A gene library of Agrobacterium tumefaciens C58 has been constructed in the plasmid vector pACYC184. A recombinant plasmid was isolated from the library by interspecific complementation in E. coli, which contained the A. tumefaciens recA gene. Heterologous Southern blotting and DNA sequence analysis have demonstrated the existence of considerable homology between the recA genes of A. tumefaciens, E. coli and R. meliloti.Abbreviations MMS methyl methanesulfonate - UV ultraviolet light - bp base pairs - kbp kilo base pairs - dATP deoxyadenosine 5-triphosphate - dNTP deoxynucleoside triphosphate - Ap ampicillin - Cm chloramphenicol - Km kanamycin - Tet tetracycline     

The levels of repair of endonuclease III-sensitive sites, 6-4 photoproducts and cyclobutane pyrimidine dimers differ in a point mutant forRAD14, theSaccharomyces cerevisiae homologue of the human gene defective inXPA patients

In the accompanying paper we demonstrated that endonuclease III-sensitive sites in theMAT andHML loci ofSaccharomyces cerevisiae are repaired by the Nucleotide Excision Repair (NER) pathway. In the current report we investigated the repair of endonuclease III sites, 6-4 photoproducts and cyclobutane pyrimidine dimers (CPDs) in arad14-2 point mutant and in arad14 deletion mutant. TheRAD14 gene is the yeast homologue of the human gene that complements the defect in cells from xeroderma pigmentosum (XP) patients belonging to complementation group A. In the point mutant we observed normal repair of endonuclease III sites (i.e. as wild type), but no removal of CPDs at theMAT andHML loci. Similar experiments were undertaken using the recently createdrad14 deletion mutant. Here, neither endonuclease III sites nor CPDs were repaired inMAT a orHMR a. Thus the point mutant appears to produce a gene product that permits the repair of endonuclease III sites, but prevents the repair of CPDs. Previously it was found that, in the genome overall, repair of 6-4 photoproducts was less impaired than repair of CPDs in the point mutant. The deletion mutant repairs neither CPDs nor 6-4 photoproducts in the genome overall. This finding is consistent with the RAD14 protein being involved in lesion recognition in yeast. A logical interpretation is that therad14-2 point mutant produces a modified protein that enables the cell to repair endonuclease III sites and 6-4 photoproducts much more efficiently than CPDs. This modified protein may aid studies designed to elucidate the role of the RAD14 protein in lesion recognition.     

Expression of the recA gene of Escherichia coli in several species of gram-negative bacteria

Summary A broad host range plasmid containing an operon fusion between the recA and lacZ genes of Escherichia coli was introduced into various aerobic and facultative gram-negative bacteria — 30 species belonging to 20 different genera — to study the expression of the recA gene after DNA damage. These included species of the families Enterobacteriaceae, Pseudomonadaceae, Rhizobiaceae, Vibrionaceae, Neisseriaceae, Rhodospirillaceae and Azotobacteraceae. Results obtained show that all bacteria tested, except Xanthomonas campestris and those of the genus Rhodobacter, are able to repress and induce the recA gene of E. coli in the absence and in the presence of DNA damage, respectively. All these data indicate that the SOS system is present in bacterial species of several families and that the LexA-binding site must be very conserved in them.     

Cloning and characterization of the recA gene of Aquaspirillum magnetotacticum

The recA gene of Aquaspirillum magnetotacticum has been isolated from a genomic library and introduced into a recA mutant strain of Escherichia coli K12. The cloned gene complemented both the recombination and DNA repair deficiency of the host and its protein product promoted the proteolytic cleavage of the LexA protein. A protein whose molecular weight is similar to that of the RecA protein of E. coli was associated with the cloned sequence.This paper is affectionately dedicated to Prof. John L. Ingraham     

Functional substitution of the recE gene of Bacillus subtilis by the recA gene of Proteus mirabilis

Summary Rec mutants of Bacillus subtilis have been tested for complementation by the recA gene of Proteus mirabilis (recApm) which was introduced into B. subtilis via the plasmid pHP334. In the recE4 mutant of B. subtilis the plasmid pHP334 restored significantly the defects in RecE functions tested: UV-sensitivity, homologous recombination (transduction and transformation) and prophage induction.Although serological methods to detect the presence of RecApm protein in B. subtilis have been unsuccessful, our results strongly indicate that the recE function of B. subtilis is analogous to the recA function of P. mirabilis.Abbreviations Cm^r resistance to chloramphenicol - Em^r resistance to erythromycin - Tc^r resistance to tetracycline - SDS sodium dodecyl sulfate - UV ultraviolet - AS ammonium sulfate     

Plasmid multimerization is dependent on RAD52 activity in Saccharomyces cerevisiae

Summary A mutant plasmid, pX, derived from the 1453 base pair small plasmid, YARp1 (or TRP1 RI circle), consists of 849 base pairs of DNA bearing the TRP1 gene and the ARS1 sequence of Saccharomyces cerevisiae and, unlike YARp1 and other commonly used yeast plasmids, highly multimerizes in a S. cerevisiae host. The multimerization of pX was dependent on RAD52, which is known to be necessary for homologous recombination in S. cerevisiae. Based upon this observation, a regulated system of multimerization of pX with GAL1 promoter-driven RAD52 has been developed. We conclude that the regulated multimerization of pX could provide a useful model system to study genetic recombination in the eukaryotic cell, in particular to investigate recombination intermediates and the effects of various trans-acting mutations on the multimerization and recombination of plasmids.     

青枯雷尔氏菌胞外多糖合成缺失突变株构建及其生物学特性

【目的】由青枯雷尔氏菌(Ralstonia solanacearum)引起的植物青枯病是一种毁灭性土传病害。胞外多糖(extracellular polysaccharides,EPS)是青枯雷尔氏菌关键的致病因子之一。通过构建胞外多糖缺失突变株,研究胞外多糖在青枯病致病中的作用。【方法】从青枯雷尔氏菌FJAT-91的基因组中克隆出胞外多糖合成结构基因epsD同源臂,克隆至自杀性质粒p K18mobsacB,再将庆大霉素抗性基因(Gm)插入同源臂中间,获得重组质粒p K18-epsD。将重组质粒转化至青枯雷尔氏菌FJAT-91感受态细胞中,通过同源重组敲除epsD基因,获得EPS合成缺失的突变株FJAT-91Δeps 。研究突变株与野生菌株在菌落形态、胞外多糖合成、运动能力、定殖能力的差异性。【结果】突变菌株FJAT-91ΔepsD与出发菌株FJAT-91相比:胞外多糖产量显著减少,生长较慢;泳动能力(swimming motility)和群集运动能力(swarming motility)显著降低;在番茄苗根部和茎部的定殖能力显著降低;弱化指数(AI)为0.905,鉴定为无致病力菌株。【结论】胞外多糖在青枯雷尔氏菌的致病中起着关键的作用,本课题研究成果为开发植物疫苗提供了优良的材料与研究基础。     

Repair of UV-irradiated plasmid DNA in a Saccharomyces cerevisiae rad3 mutant deficient in excision-repair of pyrimidine dimers

Summary The repair of UV-irradiated DNA of plasmid pBB29 was studied in an incision-defective rad3-2 strain of Saccharomyces cerevisiae and in a uvrA6 strain of Escherichia coli by the measurement of cell transformation. Plasmid pBB29 used in these experiments contained as markers the DNA of nuclear yeast gene LEU-2 and DNA of the bacterial plasmid pBR327 with resistance to Tet and Amp enabling simultaneous screening of transformant cells in both microorganisms.We found that the yeast rad3-2 mutant, deficient in incision of UV-induced pyrimidine dimers in nuclear DNA, was fully capable of repairing such lessions in plasmid DNA. The repair efficiency was comparable to that of the wild-type cells. The E. coli uvrA6 mutant, deficient in a specific nuclease for pyrimidine dimer excision from chromosomal DNA, was unable to repair UV-damaged plasmid DNA. The difference in repair capacity between the uvrA6 mutant strain and the wild-type strain was of several thousand-fold.It seems that the rad3 mutation, which confers deficiency in the DNA excision-repair system in yeast, is limited only to the nuclear DNA.