UV stimulation of chromosomal marker exchange in Sulfolobus acidocaldarius: implications for DNA repair, conjugation and homologous recombination at extremely high temperatures

The hyperthermophilic archaeon Sulfolobus acidocaldarius exchanges and recombines chromosomal markers by a conjugational mechanism, and the overall yield of recombinants is greatly increased by previous exposure to UV light. This stimulation was studied in an effort to clarify its mechanism and that of marker exchange itself. A variety of experiments failed to identify a significant effect of UV irradiation on the frequency of cell pairing, indicating that subsequent steps are primarily affected, i.e., transfer of DNA between cells or homologous recombination. The UV-induced stimulation decayed rather quickly in parental cells during preincubation at 75 degrees, and the rate of decay depended on the incubation temperature. Preincubation at 75 degrees decreased the yield of recombinants neither from unirradiated parental cells nor from parental suspensions subsequently irradiated. We interpret these results as evidence that marker exchange is stimulated by recombinogenic DNA lesions formed as intermediates in the process of repairing UV photoproducts in the S. acidocaldarius chromosome.     

Rates of spontaneous mutation in an archaeon from geothermal environments.

To estimate the efficacy of mechanisms which may prevent or repair thermal damage to DNA in thermophilic archaea, a quantitative assay of forward mutation at extremely high temperature was developed for Sulfolobus acidocaldarius, based on the selection of pyrimidine-requiring mutants resistant to 5-fluoro-orotic acid. Maximum-likelihood analysis of spontaneous mutant distributions in wild-type cultures yielded maximal estimates of (2.8 +/- 0.7) x 10(-7) and (1.5 +/- 0.6) x 10(-7) mutational events per cell per division cycle for the pyrE and pyrF loci, respectively. To our knowledge, these results provide the first accurate measurement of the genetic fidelity maintained by archaea that populate geothermal environments. The measured rates of forward mutation at the pyrE and pyrF loci in S. acidocaldarius are close to corresponding rates reported for protein-encoding genes of Escherichia coli. The normal rate of spontaneous mutation in E. coli at 37 degrees C is known to require the functioning of several enzyme systems that repair spontaneous damage in DNA. Our results provide indirect evidence that S. acidocaldarius has cellular mechanisms, as yet unidentified, which effectively compensate for the higher chemical instability of DNA at the temperatures and pHs that prevail within growing Sulfolobus cells.     

Identification of Weissella species by the genus-specific amplified ribosomal DNA restriction analysis

To investigate the generality of efficient double-strand break repair and damage-induced mutagenesis in hyperthermophilic archaea, we systematically measured the effects of five DNA-damaging agents on Sulfolobus acidocaldarius and compared the results to those obtained for Escherichia coli under corresponding conditions. The observed lethality of gamma-radiation was very similar for S. acidocaldarius and E. coli, arguing against unusually efficient double-strand break repair in S. acidocaldarius. In addition, DNA-strand-breaking agents (gamma-radiation or bleomycin), as well as DNA-cross-linking agents (mechlorethamine, butadiene diepoxide or cisplatin) stimulated forward mutation, reverse mutation, and formation of recombinants via conjugation in Sulfolobus cells. Although two of the five DNA-damaging agents failed to revert the E. coli auxotrophs under these conditions, all five reverted S. acidocaldarius auxotrophs.     

Recombination of synthetic oligonucleotides with prokaryotic chromosomes: substrate requirements of the Escherichia coli/lambdaRed and Sulfolobus acidocaldarius recombination systems

In order to reveal functional properties of recombination involving short ssDNAs in hyperthermophilic archaea, we evaluated oligonucleotide-mediated transformation (OMT) in Sulfolobus acidocaldarius and Escherichia coli as a function of the molecular properties of the ssDNA substrates. Unmodified ssDNAs as short as 20–22 nt yielded recombinants in both organisms, as did longer DNAs forming as few as 2–5 base pairs on one side of the genomic mutation. The two OMT systems showed similar responses to certain end modifications of the oligonucleotides, but E. coli was found to require a 5' phosphate on 5'-limited ssDNA whereas this requirement was not evident in S. acidocaldarius . The ability of both E. coli and S. acidocaldarius to incorporate short, mismatched ssDNAs into their genomes raises questions about the biological significance of this capability, including its phylogenetic distribution among microorganisms and its impact on genome stability. These questions seem particularly relevant for S. acidocaldarius , as this archaeon has natural competence for OMT, encodes no MutSL homologues and thrives under environmental conditions that accelerate DNA decomposition.     

Repair of DNA damage in shuttle vectors, virus, and chromosomal DNAs may depend on their biological imprinting--a 'Pygmalion' effect

We have created a cell line that can repair damage in chromosomal DNA and in herpes virus, while not repairing the same damage in shuttle vectors (pZ189 and pRSVcat). This cell line, a xeroderma pigmentosum (XP) revertant, repairs the minor (6-4)-photoproducts, but not cyclobutane dimers, in chromosomal DNA. The phenotype of this revertant after irradiation with ultraviolet (UV) light is the same as that of normal cells for survival, repair replication, recovery of rates of DNA and RNA synthesis, and sister-chromatid exchange formation, which indicates that a failure to mend cyclobutane dimers may be irrelevant to the fate of irradiated human cells. The two shuttle vectors were grown in Escherichia coli and assayed during transient passage in human cells, whereas the herpes virus was grown and assayed exclusively in mammalian cells. The ability of the XP revertant to distinguish between the shuttle vector and herpes virus DNA molecules according to their 'cultural background', i.e., bacterial or mammalian, may indicate that one component of the repair of UV damage involves gene products that recognize DNA markers that are uniquely mammalian, such as DNA methylation patterns. This component of excision repair may be involved in the original defect and the reversion of XP group A cells.     

Mutation of recF, recJ, recO, recQ, or recR improves Hfr recombination in resolvase-deficient ruv recG strains of Escherichia coli.

The formation of recombinants in Hfr crosses was studied in Escherichia coli strains carrying combinations of genes known to affect recombination and DNA repair. Mutations in ruv and recG eliminate activities that have been shown to process Holliday junction intermediates by nuclease cleavage and/or branch migration. Strains carrying null mutations in both ruv and recG produce few recombinants in Hfr crosses and are extremely sensitive to UV light. The introduction of additional mutations in recF, recJ, recO, recQ, or recR is shown to increase the yield of recombinants by 6- to 20-fold via a mechanism that depends on recBC. The products of these genes have been linked with the initiation of recombination. We propose that mutation of recF, recJ, recO, recQ, or recR redirects recombination to events initiated by the RecBCD enzyme. The strains constructed were also tested for sensitivity to UV light. Addition of recF, recJ, recN, recO, recQ, or recR mutations had no effect on the survival of ruv recG strains. The implications of these findings are discussed in relation to molecular models for recombination and DNA repair that invoke different roles for the branch migration activities of the RuvAB and RecG proteins.     

Stimulation of DNA repair and increased light output in response to UV irradiation in Escherichia coli expressing lux genes.

It has previously been suggested that the evolutionary drive of bacterial bioluminescence is a mechanism of DNA repair. By assessing the UV sensitivity of Escherichia coli, it is shown that the survival of UV-irradiated E. coli constitutively expressing luxABCDE in the dark is significantly better than either a strain with no lux gene expression or the same strain expressing only luciferase (luxAB) genes. This shows that UV resistance is dependent on light output, and not merely on luciferase production. Also, bacterial survival was found to be dependent on the conditions following UV irradiation, as bioluminescence-mediated repair was not as efficient as repair in visible light. Moreover, photon emission revealed a dose-dependent increase in light output per cell after UV exposure, suggesting that increased lux gene expression correlates with UV-induced DNA damage. This phenomenon has been previously documented in organisms where the lux genes are under their natural luxR regulation but has not previously been demonstrated under the regulation of a constitutive promoter.     

Detection of DNA damage in prokaryotes by terminal deoxyribonucleotide transferase-mediated dUTP nick end labeling

Numerous agents can damage the DNA of prokaryotes in the environment (e.g., reactive oxygen species, irradiation, and secondary metabolites such as antibiotics, enzymes, starvation, etc.). The large number of potential DNA-damaging agents, as well as their diverse modes of action, precludes a simple test of DNA damage based on detection of nucleic acid breakdown products. In this study, free 3'-OH DNA ends, produced by either direct damage or excision DNA repair, were used to assess DNA damage. Terminal deoxyribonucleotide transferase (TdT)-mediated dUTP nick end labeling (TUNEL) is a procedure in which 3'-OH DNA ends are enzymatically labeled with dUTP-fluorescein isothiocyanate using TdT. Cells labeled by this method can be detected using fluorescence microscopy or flow cytometry. TUNEL was used to measure hydrogen peroxide-induced DNA damage in the archaeon Haloferax volcanii and the bacterium Escherichia coli. DNA repair systems were implicated in the hydrogen peroxide-dependent generation of 3'-OH DNA ends by the finding that the protein synthesis inhibitors chloramphenicol and diphtheria toxin blocked TUNEL labeling of E. coli and H. volcanii, respectively. DNA damage induced by UV light and bacteriophage infection was also measured using TUNEL. This methodology should be useful in applications where DNA damage and repair are of interest, including mutant screening and monitoring of DNA damage in the environment.     

2'-deoxyribonolactone lesion produces G->A transitions in Escherichia coli

2'-deoxyribonolactone (dL) is a C1'-oxidized abasic site damage generated by a radical attack on DNA. Numerous genotoxic agents have been shown to produce dL including UV and gamma-irradiation, ene-dye antibiotics etc. At present the biological consequences of dL present in DNA have been poorly documented, mainly due to the lack of method for introducing the lesion in oligonucleotides. We have recently designed a synthesis of dL which allowed investigation of the mutagenicity of dL in Escherichia coli by using a genetic reversion assay. The lesion was site-specifically incorporated in a double-stranded bacteriophage vector M13G*1, which detects single-base-pair substitutions at position 141 of the lacZalpha gene by a change in plaque color. In E.coli JM105 the dL-induced reversion frequency was 4.7 x 10(-5), similar to that of the classic abasic site 2'-deoxyribose (dR). Here we report that a dL residue in a duplex DNA codes mainly for thymidine. The processing of dL in vivo was investigated by measuring lesion-induced mutation frequencies in DNA repair deficient E.coli strains. We showed a 32-fold increase in dL-induced reversion rate in AP endonuclease deficient (xth nfo) mutant compared with wild-type strain, indicating that the Xth and Nfo AP endonucleases participate in dL repair in vivo.     

Exchange of genetic markers at extremely high temperatures in the archaeon Sulfolobus acidocaldarius.

When cells of two auxotrophic mutants of Sulfolobus acidocaldarius are mixed and incubated on solid medium, they form stable genetic recombinants which can be selected, enumerated, and characterized. Any of a variety of auxotrophic markers can recombine with each other, and the phenomenon has been observed at temperatures of up to 84 degrees C. The ability to exchange and recombine chromosomal markers appears to be an intrinsic property of S. acidocaldarius strains. It occurs between two cell lines derived from the same parent or from different parents and also between a recombinant and its parent. This is the first observation of chromosomal marker exchange in archaea from geothermal environments and provides the first functional evidence of generalized, homologous recombination at such high temperatures.     

Overlapping functions of recD, recJ and recN provide evidence of three epistatic groups of genes in Escherichia coli recombination and DNA repair

The recD, recJ and recN genes of Escherichia coli K-12 have been shown to be involved in genetic recombination and DNA repair in this organism. Yet, mutation of any one of these genes does not seem to interfere much with the recovery of recombinants from conjugational crosses. Strains carrying all possible combinations of mutations inactivating these genes were constructed and examined for their recombination proficiency and sensitivity to UV light. The recD recJ and recJ recN double mutants are moderately sensitive to UV light and slightly deficient in recombination. A combination of mutations in all 3 genes produced strains that are very deficient in recombination (50- to 100-fold reduction) and strikingly sensitive to UV light. We conclude that these genes provide overlapping activities that compensate for one another in the single mutants. On the basis of these and other data, recombination genes are classified into 3 epistatic groups that define activities which function pre-synaptically or post-synaptically to promote genetic exchanges catalysed by RecA.     

Repair of DNA damage in shuttle vectors,virus, and chromosomal DNAs may depend on their biological imprinting — a ‘Pygmalion’ effect

We have created a cell line that can repair damage in chromosomal DNA and in herpes virus, while not repairing the same damage in shuttle vectors (pZ189 and pRSVcat). This cell line, a xeroderma pigmentosum (XP) revertant, repairs the minor (6-4)-photoproducts, but not cyclobutane dimers, in chromosomal DNA. The phenotype of this revertant after irradiation with ultraviolet (UV) light is the same as that of normal cells for survival, repair replication, recovery of rates of DNA and RNA synthesis, and sister-chromatid exchange formation, which indicates that a failure to mend cyclobutane dimers may be irrelevant to the fate of irradiated human cells. The two shuttle vectors were grown in Escherichia coli and assayed during transient passage in human cells, whereas the herpes virus was grown and assayed exclusively in mammalian cells. The ability of the XP revertant to distinguish between the shuttle vector and herpes virus DNA molecules according to their ‘cultural background’, i.e., bacterial or mammalian, may indicate that one component of the repair of UV damage involves gene products that recognize DNA markers that are uniquely mammalian, such as DNA methylation patterns. This component of excision repair may be involved in the original defect and the reversion of XP group A cells.     

Differential repair of DNA damage in specific nucleotide sequences in monkey cells.

An immunological method was developed that isolates DNA fragments containing bromouracil in repair patches from unrepaired DNA using a monoclonal antibody that recognizes bromouracil. Cultured monkey cells were exposed to either UV light or the activated carcinogen aflatoxin B1 and excision repair of damage in DNA fragments containing the integrated and transcribed E. coli gpt gene was compared to that in the genome overall. A more rapid repair, of both UV and AFB1 damage was observed in the DNA fragments containing the E. coli gpt genes. The more efficient repair of UV damage was not due to a difference in the initial level of pyrimidine dimers as determined with a specific UV endonuclease. Consistent with previous observations using different methodology, repair of UV damage in the alpha sequences was found to occur at the same rate as that in the genome overall, while repair of AFB1 damage was deficient in alpha DNA. The preferential repair of damage in the gpt gene may be related to the functional state of the sequence and/or to alterations produced in the chromatin conformation by the integration of plasmid sequences carrying the gene.     

recA gene of Escherichia coli complements defects in DNA repair and mutagenesis in Streptomyces fradiae JS6 (mcr-6).

Streptomyces fradiae JS6 (mcr-6) is a mutant which is defective in repair of DNA damage induced by a variety of chemical mutagens and UV light. JS6 is also defective in error-prone (mutagenic) DNA repair (J. Stonesifer and R. H. Baltz, Proc. Natl. Acad. Sci. USA 82:1180-1183, 1985). The recA gene of Escherichia coli, cloned in a bifunctional vector that replicates in E. coli and Streptomyces spp., complemented the mutation in S. fradiae JS6, indicating that E. coli and S. fradiae express similar SOS responses and that the mcr+ gene product of S. fradiae is functionally analogous to the protein encoded by the recA gene of E. coli.     

Reciprocal and non-reciprocal homologous recombination between Escherichia coli chromosomal DNA and ultraviolet light-irradiated plasmid DNA

Plasmid DNA substrates were used to study ultraviolet (UV)-induced recombination events in Escherichia coli host cells. Plasmids derived from pBR322, containing all or part of the lac operon of E. coli, were irradiated with ultraviolet light before transformation into E. coli strains of different recA and lacY genotypes. Recombinational exchanges were identified by phenotypic changes in lactose utilization and were confirmed by restriction analysis of isolated plasmids. Ultraviolet-induced reciprocal plasmid-chromosome recombination occurred at a slightly higher frequency then non-reciprocal chromosome-to-plasmid recombination, and at a much higher frequency than non-reciprocal plasmid-to-chromosome recombination. These frequencies did not depend on segregative mechanisms. The asymmetry of non-reciprocal exchange was not due to the particular arrangement of wild-type and lacY1 alleles because the same results were observed when these were interchanged. The host recA gene was required for plasmid-chromosome recombination, and slightly enhanced plasmid survival. Evidence for plasmid replication prior to recombination was found in reciprocal recombinants, but rarely in the non-reciprocal recombinants analyzed. Irradiation of competent bacterial host cells prior to transformation did not effectively induce plasmid-chromosome recombination.     

Diverse roles for histone H2A modifications in DNA damage response pathways in yeast

There are many types of DNA damage that are repaired by a multiplicity of different repair pathways. All damage and repair occur in the context of chromatin, and histone modifications are involved in many repair processes. We have analyzed the roles of H2A and its modifications in repair by mutagenizing modifiable residues in the N- and C-terminal tails of yeast H2A and by testing strains containing these mutations in multiple DNA repair assays. We show that residues in both tails are important for homologous recombination and nonhomologous end-joining pathways of double-strand break repair, as well as for survival of UV irradiation and oxidative damage. We show that H2A serine 122 is important for repair and/or survival in each of these assays. We also observe a complex pattern of H2A phosphorylation at residues S122, T126, and S129 in response to different damage conditions. We find that overlapping but nonidentical groups of H2A residues in both tails are involved in different pathways of repair. These data suggest the presence of a set of H2A "damage codes" in which distinct patterns of modifications on both tails of H2A may be used to identify specific types of damage or to promote specific repair pathways.     

Effect of SOS-induced Pol II, Pol IV, and Pol V DNA polymerases on UV-induced mutagenesis and MFD repair in Escherichia coli cells

Irradiation of organisms with UV light produces genotoxic and mutagenic lesions in DNA. Replication through these lesions (translesion DNA synthesis, TSL) in Escherichia coli requires polymerase V (Pol V) and polymerase III (Pol III) holoenzyme. However, some evidence indicates that in the absence of Pol V, and with Pol III inactivated in its proofreading activity by the mutD5 mutation, efficient TSL takes place. The aim of this work was to estimate the involvement of SOS-inducible DNA polymerases, Pol II, Pol IV and Pol V, in UV mutagenesis and in mutation frequency decline (MFD), a mechanism of repair of UV-induced damage to DNA under conditions of arrested protein synthesis. Using the argE3-->Arg(+) reversion to prototrophy system in E. coli AB1157, we found that the umuDC-encoded Pol V is the only SOS-inducible polymerase required for UV mutagenesis, since in its absence the level of Arg(+) revertants is extremely low and independent of Pol II and/or Pol IV. The low level of UV-induced Arg(+) revertants observed in the AB1157mutD5DumuDC strain indicates that under conditions of disturbed proofreading activity of Pol III and lack of Pol V, UV-induced lesions are bypassed without inducing mutations. The presented results also indicate that Pol V may provide substrates for MFD repair; moreover, we suggest that only those DNA lesions which result from umuDC-directed UV mutagenesis are subject to MFD repair.