RESUMO
Chromosome replication in Escherichia coli is normally initiated at oriC, the origin of chromosome replication. E. coli cells possess at least three additional initiation systems for chromosome replication that are normally repressed but can be activated under certain specific conditions. These are termed the stable DNA replication systems. Inducible stable DNA replication (iSDR), which is activated by SOS induction, is proposed to be initiated from a D-loop, an early intermediate in homologous recombination. Thus, iSDR is a form of recombination-dependent DNA replication (RDR). Analysis of iSDR and RDR has led to the proposal that homologous recombination and double-strand break repair involve extensive semiconservative DNA replication. RDR is proposed to play crucial roles in homologous recombination, double-strand break repair, restoration of collapsed replication forks, and adaptive mutation. Constitutive stable DNA replication (cSDR) is activated in mhA mutants deficient in RNase HI or in recG mutants deficient in RecG helicase. cSDR is proposed to be initiated from an R-loop that can be formed by the invasion of duplex DNA by an RNA transcript, which most probably is catalyzed by RecA protein. The third form of SDR is nSDR, which can be transiently activated in wild-type cells when rapidly growing cells enter the stationary phase. This article describes the characteristics of these alternative DNA replication forms and reviews evidence that has led to the formulation of the proposed models for SDR initiation mechanisms. The possible interplay between DNA replication, homologous recombination, DNA repair, and transcription is explored.
Assuntos
Replicação do DNA , Modelos Genéticos , Recombinação Genética , Transcrição Gênica , Bacteriófago T4/genética , Dano ao DNA , Reparo do DNA , DNA Mitocondrial/genética , Escherichia coli/genética , Conformação de Ácido Nucleico , Receptores de Antígenos/genética , Leveduras/genéticaRESUMO
rnh mutants harboring pBR322 were found to contain several slowly migrating DNA species when examined by agarose gel electrophoresis. The plasmid DNA from rnh mutants included large molecules, i.e. plasmids two, three or four times the size of a single plasmid unit. That this DNA contained concatemeric plasmid joined in a head-to-tail fashion was determined by digestion with restriction endonucleases that cleaved the monomeric plasmid DNA at a unique site. This treatment resulted in migration of the plasmid DNA at a mobility identical to that of linearized monomeric plasmid by agarose gel electrophoresis. This was confirmed by electron microscopy. Plasmid concatemer formation was detected with several high-copy-number (relaxed type) plasmids but not with low-copy-number (stringent) plasmids. Concatemer formation was dependent on RecA+ and RecF+ functions since several recA and recF mutations abolished concatemer formation. ColE1-type plasmids were previously shown to replicate in rnh mutants in the absence of DNA polymerase I (PolI) activity. This DNA PolI-independent plasmid replication was also examined for its dependence on the recF and recA gene products. rnh- polA(Ts) recF- strains were efficiently transformed with these plasmids at 30 degrees C and 42 degrees C, indicating the presence of DNA PolI-independent replication under recF- conditions. The presence or absence of plasmid replication in rnh- polA- recA(Ts) strains was also examined by measuring the increase in total amounts of plasmid. The results indicated that DNA PolI-independent replication occurred in these triple mutants at 42 degrees C as well as at 30 degrees C. It was concluded that the recombination event giving rise to concatemer formation was not essential for DNA PolI-independent replication in rnh mutants.
Assuntos
Endorribonucleases/metabolismo , Escherichia coli/enzimologia , Mutação , Plasmídeos , Proteínas de Bactérias/genética , DNA Polimerase I/metabolismo , Replicação do DNA , DNA Bacteriano , Eletroforese em Gel de Ágar , Escherichia coli/genética , Conformação de Ácido Nucleico , Recombinases Rec A/genética , Ribonuclease HRESUMO
When Escherichia coli cells are subjected to genetic stress by exposure to agents or conditions that transiently block DNA replication, the mode of DNA replication is profoundly altered. One of the alterations is the induction of inducible stable DNA replication (iSDR) that does not require the initiator protein, DnaA, and occurs despite the presence of rifampin and chloramphenicol, which inhibit the initiation of usual chromosome replication at oriC. It has been demonstrated that iSDR starts primarily from both the oriC and terC regions of the chromosome. To precisely map the iSDR origin (oriM1) located in the oriC region, various oriC fragments were inserted into a plasmid vector derived from pSC101, and the copy number of these plasmid constructs was measured in the presence of rifampin and chloramphenicol after cells were induced for the SOS response by thymine starvation. The results indicated that there are at least two origins for iSDR within the minimal oriC; one (oriM1A) is located between the BamHI (coordinate +1) and the AvaII(155) sites, and the other (oriM1B) between the AvaII(155) and the HindIII(244) sites. Furthermore, a 263 bp fragment containing oriM1, which was placed at the att lambda site of the chromosome, was found to initiate chromosome replication in the presence of the drugs when cells were starved of thymine. Introduction of additional copies of oriM1 into a cell stimulated initiation of iSDR at oriM1 on the chromosome. The result supported the model that iSDR starts from D-loops created between oriM1 sequences and that the amount of D-loops determines the level of the iSDR activity.
Assuntos
Cromossomos Bacterianos , Dano ao DNA , Replicação do DNA/genética , Escherichia coli/genética , Resposta SOS em Genética , Proteínas de Bactérias/metabolismo , Southern Blotting , Cloranfenicol/farmacologia , Replicação do DNA/efeitos dos fármacos , DNA Bacteriano/análise , DNA Bacteriano/biossíntese , Proteínas de Ligação a DNA/metabolismo , Escherichia coli/efeitos dos fármacos , Escherichia coli/metabolismo , Vetores Genéticos , Genótipo , Cinética , Plasmídeos , Mapeamento por Restrição , Rifampina/farmacologia , Timina/metabolismo , Fatores de TempoRESUMO
In stable DNA replication (sdrA/rnh) mutants of Escherichia coli, initiation of rounds of DNA replication occurs in the absence of the normal origin of replication, oriC. To determine whether or not the initiation occurs at a fixed site(s) on the chromosome in sdrA mutants, the DNA from exponentially growing sdrA mutant cells with or without the oriC site (delta oriC) was analyzed for the relative copy numbers of various genes along the chromosome. The results suggest that there are at least four fixed sites or regions of the sdrA delta oriC chromosome from which DNA replication can be initiated in the absence of the oriC sequence.
Assuntos
Replicação do DNA , DNA Bacteriano/genética , Escherichia coli/genética , Mutação , Mapeamento Cromossômico , Cromossomos Bacterianos , Escherichia coli/ultraestrutura , Marcadores Genéticos , PlasmídeosRESUMO
Induction of the SOS response in Escherichia coli activates normally repressed DNA replication which is termed inducible stable DNA replication (iSDR). We previously demonstrated that initiation of iSDR requires the products of genes, such as recA, recB and recC, that are involved in the early stages of homologous recombination. By measuring the copy number increase of the origin (oriM1) region on the chromosome, we show, in this study, that initiation of iSDR is stimulated by mutations in the ruvA, ruvC and recG genes which are involved in the late stages of homologous recombination. Continuation of iSDR, on the other hand, is inhibited by these mutations. The results suggest that Holliday recombination intermediates, left on the chromosome due to abortive recombination, arrest replication fork movement. Low levels of iSDR and sfiA (sulA) gene expression were also observed in exponentially growing ruvA, ruvC and recG mutants, suggesting that the SOS response is chronically induced in these mutants. We propose that replication forks are arrested in these mutants, albeit at a low frequency, even under the normal (uninduced) conditions.
Assuntos
Proteínas de Bactérias/fisiologia , Cromossomos Bacterianos , Dano ao DNA , DNA Helicases/fisiologia , Replicação do DNA , DNA Bacteriano/biossíntese , Proteínas de Ligação a DNA/fisiologia , Endodesoxirribonucleases/fisiologia , Proteínas de Escherichia coli , Escherichia coli/genética , Resposta SOS em Genética/genética , Proteínas de Bactérias/genética , DNA Helicases/genética , DNA Bacteriano/genética , Proteínas de Ligação a DNA/genética , Endodesoxirribonucleases/genética , Recombinação GenéticaRESUMO
The mechanism of recA polA lethality in Escherichia coli has been studied. Complementation tests have indicated that both the 5'-->3' exonuclease and the polymerization activities of DNA polymerase I are essential for viability in the absence of RecA protein, whereas the viability and DNA replication of DNA polymerase I-defective cells depend on the recombinase activity of RecA. An alkaline sucrose gradient sedimentation analysis has indicated that RecA has only a minor role in Okazaki fragment processing. Double-strand break repair is proposed for the major role of RecA in the absence of DNA polymerase I. The lexA(Def)::Tn5 mutation has previously been shown to suppress the temperature-sensitive growth of recA200(Ts) polA25::spc mutants. The lexA(Def) mutation can alleviate impaired DNA synthesis in the recA200(Ts) polA25::spc mutant cells at the restrictive temperature. recF+ is essential for this suppression pathway. recJ and recQ mutations have minor but significant adverse effects on the suppression. The recA200(Ts) allele in the recA200(Ts) polA25::spc lexA(Def) mutant can be replaced by delta recA, indicating that the lexA(Def)-induced suppression is RecA independent. lexA(Def) reduces the sensitivity of delta recA polA25::spc cells to UV damage by approximately 10(4)-fold. lexA(Def) also restores P1 transduction proficiency to the delta recA polA25::spc mutant to a level that is 7.3% of the recA+ wild type. These results suggest that lexA(Def) activates a RecA-independent, RecF-dependent recombination repair pathway that suppresses the defect in DNA replication in recA polA double mutants.
Assuntos
Reparo do DNA , Escherichia coli/genética , Genes Letais , Integrases , Recombinação Genética , Serina Endopeptidases , Proteínas de Bactérias/genética , DNA Nucleotidiltransferases/metabolismo , DNA Polimerase I/metabolismo , Replicação do DNA , Mutação , Biossíntese de Proteínas , Recombinases Rec A/genética , Recombinases Rec A/metabolismo , Recombinases , Regulon , TemperaturaRESUMO
rnhA224 and rnhA339::cat mutants which lack RNase HI activity were found to constitutively express the sfiA::lacZ operon fusion in a recA+ lexA(+)-dependent manner. The sfiA::lacZ expression (indicating SOS induction) in rnhA mutants was increased to higher levels by the introduction of the recD1903 or recB21 mutation. The SOS induction in these cells was further enhanced by nutritional shift up from casamino acid medium to Luria broth. Although the extent by which the recD and recB mutations increased the sfiA expression in rnhA mutants was similar, the rnhA224 recB21 double mutant had plating efficiencies that were 25-fold lower on casamino acid plates and 5 x 10(5)-fold lower on Luria broth plates than the respective plating efficiencies of either rnhA224 recD or rnhA::cat recD double mutants. Whereas the recD mutation inactivates the exonuclease activity of the RecBCD (Exo V) enzyme without reducing the recombination proficiency of the mutant, the recB21 mutation abolishes both the exonuclease activity and recombination capability. Therefore, in the absence of both RNase HI and Exo V activities, homologous recombination functions become crucial for viability, particularly in Luria broth. Introduction of mutations in recA, recJ and recN exacerbated the phenotypes. It is proposed that R-loops which persist due to the lack of RNase HI activity can be removed by two alternative routes of DNA repair: one involving Exo V, Exo I and DNA polymerase I, and the other involving both the RecBCD and RecF pathways of homologous recombination activities. The isolation of RNA polymerase mutants that constitutively express the SOS response at high levels and exhibit remarkable broth-sensitivity lend strong support to the contention that increased amounts of the persisting R-loop in rnhA mutants growing in Luria broth give rise to a stronger SOS response.
Assuntos
DNA Helicases/metabolismo , Proteínas de Escherichia coli , Escherichia coli/enzimologia , Exodesoxirribonucleases/metabolismo , Mutação , Recombinação Genética , Ribonuclease H/metabolismo , Resposta SOS em Genética , DNA Bacteriano/biossíntese , RNA Polimerases Dirigidas por DNA/genética , Exodesoxirribonuclease VAssuntos
Cromossomos Bacterianos , Replicação do DNA/efeitos dos fármacos , DNA Bacteriano/biossíntese , Escherichia coli/metabolismo , Isótopos de Carbono , Centrifugação com Gradiente de Concentração , Cloranfenicol/farmacologia , Genes Letais , Hibridização Genética , Mutação , Ácido Nalidíxico/farmacologia , Temperatura , Moldes Genéticos , Timidina/metabolismo , Timina/farmacologia , TrítioAssuntos
Replicação do DNA , DNA Bacteriano/biossíntese , Aminoglicosídeos , Antibacterianos/farmacologia , Proteínas de Bactérias/biossíntese , Cloranfenicol/farmacologia , Citarabina/farmacologia , Replicação do DNA/efeitos dos fármacos , Escherichia coli , Temperatura Alta , Hidroxiureia/farmacologia , Modelos Biológicos , Mutação , Ácido Nalidíxico/farmacologia , Biossíntese de Proteínas/efeitos dos fármacos , RNA Bacteriano/biossíntese , Timina/metabolismoRESUMO
Escherichia coli rnhA mutants lacking RNase HI chronically express the SOS response (T. Kogoma, X. Hong, G. W. Cadwell, K. G. Barnard, and T. Asai, Biochimie 75:89-99, 1993). Seventeen rpoB (Rifr) mutant alleles, which encode altered beta subunits of RNA polymerase, giving rise to resistance to rifampin, were screened for the ability to enhance or diminish constitutive expression of the SOS response in rnhA mutants. Two mutations, rpoB3595 and rpoB2, were found to enhance the SOS response 5- and 2.5-fold, respectively, only when RNase HI is absent. These mutations rendered rnhA mutant cells very sensitive to broth; i.e., the plating efficiency of the double mutants was drastically reduced when tested on broth plates. Two mutations, rpoB8 and rpoB3406, were found to diminish constitutive SOS expression in rnhA mutants by 43 and 30%, respectively. It was suggested that RNA polymerase may have a property that influences the size of DNA-RNA hybrids, the frequency of their formation, or both and that the property resides at least in part in the beta subunit of the polymerase.
Assuntos
RNA Polimerases Dirigidas por DNA/metabolismo , Escherichia coli/enzimologia , Escherichia coli/genética , Ribonuclease H/metabolismo , Resposta SOS em Genética , Deleção de Sequência , Alelos , RNA Polimerases Dirigidas por DNA/genética , Resistência Microbiana a Medicamentos/fisiologia , Escherichia coli/crescimento & desenvolvimento , Regulação Bacteriana da Expressão Gênica , Genes Bacterianos , Genótipo , Substâncias Macromoleculares , Proteínas Recombinantes de Fusão/metabolismo , Ribonuclease H/genética , Rifampina/toxicidade , beta-Galactosidase/metabolismoRESUMO
rnh (formerly termed sdrA) mutants of Escherichia coli K-12, capable of continuous DNA replication in the absence of protein synthesis (stable DNA replication), are devoid of ribonuclease H (RNase H, EC 3.1.26.4) activity. Plasmid pBR322 was found to replicate in rnh mutants in the absence of DNA polymerase I, the polA gene product, which is normally required for replication of this plasmid. The plasmid copy number in polA rnh double mutants was as high as in the wild-type strains. When a chimeric construct between pBR322 and pSC101 was introduced into a polA rnh double mutant, the replication of the plasmid via the pBR322 replicon was inhibited if the plasmid also carried an rnh+ gene or if the host harbored an F' plasmid carrying an rnh+ gene. Thus, DNA polymerase I-independent replication of pBR322 requires the absence of RNase H activity. This alternative mechanism requiring neither DNA polymerase I nor RNase H appears to involve a transcriptional event in the region of the normal origin of replication.
Assuntos
DNA Polimerase I/genética , Endorribonucleases/genética , Escherichia coli/genética , Plasmídeos , DNA Polimerase I/metabolismo , Replicação do DNA , Enzimas de Restrição do DNA , Endorribonucleases/metabolismo , Escherichia coli/enzimologia , Genótipo , Mutação , Ribonuclease H , Especificidade da Espécie , beta-Galactosidase/genéticaRESUMO
rnh mutations of Escherichia coli inactivating RNase H activity allow the initiation of rounds of DNA replication in the absence of protein synthesis (stable DNA replication). However, levels of RNase H did not change during or after the induction of stable DNA replication in rnh+ strains by incubation with nalidixic acid or UV irradiation.
Assuntos
Replicação do DNA , Endorribonucleases/fisiologia , Escherichia coli/metabolismo , Replicação do DNA/efeitos da radiação , Endorribonucleases/antagonistas & inibidores , Mutação , Ácido Nalidíxico/farmacologia , Ribonuclease HRESUMO
In previous studies, we found that the requirement for RecA protein in constitutive stable DNA replication (cSDR) can be bypassed by derepression of the LexA regulon and that DNA polymerase I (DNA PolI) is essential for this Rip (RecA-independent process) pathway of cSDR (Y. Cao, R. R. Rowland, and T. Kogoma, J. Bacteriol. 175:7247-7253, 1993). In this study, the role of DNA PolI in the Rip pathway was further examined. By using F' plasmids carrying different parts of the polA gene, a series of complementation tests was carried out to investigate the requirement for the three enzymatic activities, polymerization, 3'-->5' exonuclease, and 5'-->3' exonuclease activities, of DNA PolI. The result indicated that both the 5'-->3' exonuclease and polymerization activities of DNA PolI are essential for bypassing the requirement for RecA in cSDR but that the 3'-->5' exonuclease activity can be dispensed with. Complementation experiments with rat DNA Pol beta also supported the hypothesis that a nick translation activity is probably involved in cSDR in the absence of RecA. An analysis of DNA synthesis suggested that DNA PolI is involved in the initiation but not the elongation stage of cSDR. Moreover, the dnaE293(Ts) mutation was shown to render the bypass replication temperature sensitive despite the presence of active DNA PolI, suggesting that DNA PolIII is responsible for the elongation stage of the Rip pathway. A model which describes the possible roles of RecA in cSDR and the possible function of DNA PolI in the Rip pathway is proposed.
Assuntos
DNA Polimerase I/metabolismo , Replicação do DNA , DNA Bacteriano/biossíntese , Escherichia coli/genética , Escherichia coli/metabolismo , Exodesoxirribonucleases/metabolismo , Genes Bacterianos , Recombinases Rec A/metabolismo , DNA Polimerase II/metabolismo , DNA Polimerase III/metabolismo , DNA Bacteriano/química , Exodesoxirribonuclease V , Teste de Complementação Genética , Cinética , Modelos Estruturais , Mutagênese , Conformação de Ácido Nucleico , Plasmídeos , Recombinases Rec A/genética , Mapeamento por RestriçãoRESUMO
DNA damage-inducible DNA replication in SOS-induced Escherichia coli cells, termed inducible stable DNA replication (iSDR), has previously been shown to require either the RecBCD or the RecE pathway of homologous recombination for initiation. Here, we demonstrate that recB recC sbcC quadruple mutant cells are capable of iSDR induction and that a mutation in the recJ gene abolishes the inducibility. These results indicate that the RecF pathway of homologous recombination can also catalyze iSDR initiation.
Assuntos
Proteínas de Bactérias/metabolismo , Dano ao DNA/genética , Replicação do DNA , Proteínas de Ligação a DNA/metabolismo , Proteínas de Escherichia coli , Escherichia coli/genética , Recombinação Genética , Cromossomos Bacterianos , Resposta SOS em Genética , Raios Ultravioleta/efeitos adversosRESUMO
A deletion in the rpoH gene greatly increased the sensitivity of Escherichia coli sodA sodB mutants to oxidative stress. The effect of the rpoH deletion on sodA+ sodB+ cells was only marginal. Mutations in heat shock genes singly sensitized sodA sodB double mutant cells to plumbagin. sodA sodB double mutants were neither more sensitive nor more resistant to thermal stress than the wild type.
Assuntos
Deleção Cromossômica , Escherichia coli/genética , Genes Bacterianos , Proteínas de Choque Térmico/genética , Temperatura Alta , Oxigênio/farmacologia , Fator sigma/genética , Fatores de Transcrição , Escherichia coli/efeitos dos fármacos , Mutagênese Sítio-Dirigida , Superóxido Dismutase/genéticaRESUMO
Employing Bacillus cereus strain 2, we examined the fate of two chromosomes contained in vegetative cells in the course of sporulation. Cytological observations and quantitative estimation of deoxyribonucleic acid (DNA) confirmed the earlier observations that, during the course of sporulation, one of two chromosomes of the vegetative cell was incorporated into the sporangium and the other disappeared into the medium as the result of cell lysis. Log-phase cells, labeled completely with thymine-2-(14)C in the presence of deoxyadenosine, were cultured in the "cold" glucose-glutamate-glycine-salts medium, and culture samples, taken at intervals at successive generations, were subjected to sporulation in glutamate-salts medium. The percentage of radioactivity in the spores separated from each culture remained almost unchanged at nearly 50% and was independent of the number of generations of the preceding culture in the "cold" medium. This suggests that the selective incorporation into the sporangium of either the "older" or "younger" chromosome of a vegetative cell does not occur in the course of spore formation. Some examples of the selective and nonselective behavior of DNA molecules in cellular events in microorganisms are cited.
Assuntos
Bacillus cereus/crescimento & desenvolvimento , Cromossomos/metabolismo , Bacteriólise , Isótopos de Carbono , Diferenciação Celular , Divisão Celular , Meios de Cultura , DNA Bacteriano/análise , DNA Bacteriano/metabolismo , Desoxirribonucleases/farmacologia , Glucose/metabolismo , Glutamatos/metabolismo , Glicina/metabolismo , Lipase/farmacologia , Muramidase/farmacologia , Nucleosídeos/farmacologia , Esporos/crescimento & desenvolvimento , Esporos/metabolismo , Timina/metabolismoRESUMO
On the basis of the experiments carried out with rnhA224 mutants, we previously concluded that RNase HI is not essential for initiation of Escherichia coli chromosome replication at oriC (T. Kogoma, N.L. Subia, and K. von Meyenburg, Mol. Gen. Genet. 200:103-109, 1985). In light of the recent finding that rnhA224 is a UGA nonsense mutation which can be leaky in certain genetic backgrounds, we reexamined this conclusion with the use of rnhA339 (Null)::cat mutants. The possibility that recB+ is required for initiation at the alternative origins (oriKs) of replication in rnhA mutants was also tested. The results clearly indicated that RNase HI is not essential for oriC initiation and that recB+ is not required for initiation at oriK sites.