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At least 127 records · Page 7Linked to original sources
PubMed2001

Genetic identification of two distinct DNA polymerases, DnaE and PolC, that are essential for chromosomal DNA replication in Staphylococcus aureus.

We isolated and characterized temperature-sensitive mutants for two genes, dnaE and polC, that are essential for DNA replication in Staphylococcus aureus. DNA replication in these mutants had a slow-stop phenotype when the temperature was shifted to a non-permissive level. The dnaE gene encodes a homolog of the alpha-subunit of the DNA polymerase III holoenzyme, the replicase essential for chromosomal DNA replication in Escherichia coli. The polC gene encodes PolC, another catalytic subunit of DNA polymerase, which is specifically found in gram-positive bacteria. The wild-type dnaE or polC gene complemented the temperature-sensitive phenotypes of cell growth and DNA replication in the corresponding mutant. Single mutations resulting in amino-acid exchanges were identified in the dnaE and polC genes of the temperature-sensitive mutants. The results indicate that these genes encode two distinct DNA polymerases which are both essential for chromosomal DNA replication in S. aureus. The number of viable mutant cells decreased at non-permissive temperature, suggesting that inactivation of DnaE and PolC has a bactericidal effect and that these enzymes are potential targets of antibiotics.

Amino Acid Sequence↗
PubMed1984

Selective affinity chromatography of DNA polymerases with associated 3' to 5' exonuclease activities.

The use of 5'-AMP as a ligand for the affinity chromatography of DNA polymerases with intrinsic 3' to 5' exonuclease activities was investigated. The basis for this is that 5'-AMP would be expected to act as a ligand for the associated 3' to 5' exonuclease. The requirements for binding of Escherichia coli DNA polymerase I, T4 DNA polymerase, and calf thymus DNA polymerase delta, all of which have associated 3' to 5' exonuclease activities, to several commercially available 5'-AMP supports with different linkages of 5'-AMP to either agarose or cellulose were examined. The DNA polymerases which possessed 3' to 5' exonuclease activities were bound to agarose types in which the 5'-phosphoryl group and the 3'-hydroxyl group of the AMP were unsubstituted. Bound enzyme could be eluted by either an increase in ionic strength or competitive binding of nucleoside 5'-monophosphates. Magnesium was found to reinforce the binding of the enzyme to these affinity supports. DNA polymerase alpha, which does not have an associated 3' to 5' exonuclease activity, did not bind to any of these columns. These differences can be used to advantage for the purification of DNA polymerases that have associated 3' to 5' exonuclease activities, as well as a means for establishing the association of 3' to 5' exonuclease activities with DNA polymerases.

Adenosine Monophosphate↗
PubMed1992

Decreased fidelity of DNA polymerases and decreased DNA excision repair in aging mice: effects of caloric restriction.

Hepatic DNA polymerases from calorie restricted and ad libitum 26 month old C57BL/6 mice showed a decline in fidelity of nucleotide incorporation compared with weanling animals. Both alpha and beta polymerases from calorie restricted aged mice exhibited a higher level of fidelity than polymerases from ad libitum aged mice. UV-initiated unscheduled DNA synthesis was significantly higher in hepatocytes from weanling and 18 month old calorie restricted animals compared with cells from 18 month old ad libitum animals, while MMS-initiated unscheduled DNA synthesis did not differ significantly between cells from young and old or ad libitum and calorie restricted animals. These data suggest that calorie restriction could play a significant role in decreasing the age-related decline of cellular mechanisms expected to reduce the rate at which mutations accumulate during aging, and could potentially prolong the onset age of mutation-associated diseases of the elderly.

Aging↗
PubMed1995

Mitomycin C-induced distortions of DNA at minor alkylation sites.

Reductively-activated mitomycin C (MC) presents a high specificity to the 5'-CG site and to a lesser extent the 5'-GG site. However, its affinity is different for each 5'-CG site. This was evidenced by using the 3'-5' exonuclease activity of T4 DNA polymerase on a short DNA fragment exposed to MC, which was gradually activated by several Na2S2O4 additions. The time-delayed appearance of some exonuclease digestion stop sites (corresponding to MC-monofunctional adducts) suggests that MC discriminates between very fine structural variations. The feature of the stop sites suggests a good fit of MC in the DNA groove, in the case of the major alkylation sites, but not in the case of a minor 5'-TG alkylation site. Furthermore, it is evidenced by the use of the chemical probe hydroxylamine (HA) that MC-monoalkylation of 5'-CG (or 5'-GG) does not induce notable local structural disturbance of the DNA double helix, as opposed to alkylation of the 5'-TG site of minor specificity, which leads to significant local DNA distortion. This suggests that the 'in vivo' effect of MC is related, not only to amount of alkylated sites (essentially 5'-CG sites), but also to possible local DNA deformations (at minor alkylation sites).

Alkylation↗
PubMed1983

Resolution and reconstitution of the rec BC deoxyribonuclease of Escherichia coli.

The inactivation of rec BC nuclease activity and simultaneously the separation of 3 DNA-dependent ATPases and an ATP-independent DNases specific for single-stranded DNA have been observed after DEAE-cellulose chromatography of cell extracts from Escherichia coli. Two of the ATPases catalyze the strand separation of duplex DNA. Reconstitution of ATP-dependent DNase activity has been carried out by the combination of the separated enzymes.

Adenosine Triphosphatases↗
PubMed1983

A third DNA-dependent ATPase from Bacillus cereus free of ATP-dependent DNase activity.

The purification of ATP-dependent DNase from Bacillus cereus led to the isolation and characterization of a third DNA-dependent ATPase. The enzyme called ATPase III has been purified free of nuclease activity. None of the expected ATPases proved to be identical with ATP-dependent DNase-DNA-dependent ATPase. Separation of ATPase I, II and III and a DNase specific for single-stranded DNA from the same source excludes the possibility of ATP-dependent DNase being the action of a single enzyme molecule.

Adenosine Triphosphatases↗
PubMed1983

Enhanced polypeptide synthesis programmed by linear DNA fragments in cell-free extracts lacking exonuclease V.

Preparation of an in vitro coupled transcription-translation system from E. coli strains lacking exonuclease V has greatly improved the system for use with added linear DNA fragments. In fact, in extracts of these mutants linear fragments are stable for several hours. However the cell extracts show a high level of endogenous background. To avoid this complication extracts were prepared at 30 degrees C from a mutant carrying a temperature-sensitive exonuclease V. Polypeptides coded by a specific DNA region, e.g., delineated by restriction endonuclease sites, can now be easily identified.

DNA, Bacterial↗
PubMed1989

Pyrophosphate analogues in pyrophosphorolysis reaction catalyzed by DNA polymerases.

It is demonstrated here that rat liver DNA polymerase beta catalyzes the pyrophosphorolysis reaction with pyrophosphate (PPi) and its analogues. The substrate specificity of the PPi-binding site of several DNA polymerases was investigated. It was discovered that the ability of DNA polymerases to utilize PPi analogues instead of PPi in the pyrophosphorolysis reaction was markedly restricted. Only imidodiphosphate and methylenediphosphonate were demonstrated as participating in this process. Oxodiphosphonate and phosphonoformate inhibited DNA synthesis, but probably not via the interaction with the PPi-binding site of DNA polymerases.

Animals↗
PubMed1993

Effects of a single intrastrand d(GpG) platinum adduct on the strand separating activity of the Escherichia coli proteins RecB and RecA.

RecB and RecA proteins play key roles in the process of DNA recombination in Escherichia coli and both possess DNA unwinding activities which can displace short regions of duplex DNA in an ATP-dependent manner in vitro. We have examined the effect of the most abundant DNA adduct caused by the chemotherapeutic agent cis-diamminedichloroplatinum(II) on those activities. For this purpose, we have constructed a partially duplex synthetic oligonucleotide containing the intrastrand d(GpG) crosslink positioned at a specific site. We report here that both the DNA strand separating and DNA-dependent ATPase activities of the RecB protein are inhibited by the d(GpG) cis-DDP adduct. In contrast, neither the unwinding nor the ATPase activities of RecA protein appear to be perturbed by this lesion.

Base Sequence↗
PubMed1985

Substrate specificity of the DNA unwinding activity of the RecBC enzyme of Escherichia coli.

The RecBC enzyme of Escherichia coli promotes genetic recombination of phage or bacterial chromosomes. The purified enzyme travels through duplex DNA, unwinding and rewinding the DNA with the transient production of potentially recombinogenic single-stranded DNA. The studies reported here are aimed at understanding which chromosomal forms allow the entry of RecBC enzyme and hence may undergo RecBC enzyme-mediated recombination. Circular duplex molecules, whether covalently closed, nicked or containing single-stranded gaps of 10 to 774 nucleotides, are not detectably unwound by RecBC enzyme. Linear duplex molecules are readily unwound if they have a nearly flush-ended terminus whose 5' and 3' ends are offset by no more than about 25 nucleotides; molecules with longer single-stranded tails are poorly bound by RecBC enzyme and are infrequently unwound. The single-strand endonuclease activity of RecBC enzyme can slowly cleave gapped circles to produce molecules presumably capable of being unwound. These results provide an enzymatic basis for the recombinogenicity of double-stranded DNA ends established from genetic studies of RecBC enzyme and Chi sites, recognition sites for RecBC enzyme-mediated DNA strand cleavage.

Autoradiography↗
PubMed1987

Cutting of chi-like sequences by the RecBCD enzyme of Escherichia coli.

Chi, 5' G-C-T-G-G-T-G-G 3', stimulates coliphage lambda recombination mediated by the major recombination pathway of Escherichia coli, the RecBC pathway. Purified RecBCD enzyme makes a single strand endonuclease cleavage four to six nucleotides to the 3' side of the chi sequence. Three sequences similar to chi, 5' A-C-T-G-G-T-G-G 3', 5' G-T-T-G-G-T-G-G 3' and 5' G-C-T-A-G-T-G-G 3', have partial recombinational hotspot activity in genetic crosses. We report here that purified RecBCD enzyme preferentially cuts four nucleotides to the right of the two most active chi-like octamers. The degree of cutting correlated with the genetic activities of these sequences; this result indicates that these cleavages are essential to the genetic activity of chi and chi-like sequences.

Bacteriophage lambda↗
PubMed1989

Activation of Chi recombinational hotspots by RecBCD-like enzymes from enteric bacteria.

Chi sites, 5'G-C-T-G-G-T-G-G-3', enhance homologous recombination in Escherichia coli and are activated by the RecBCD enzyme. To test the ability of Chi to be activated by analogous enzymes from other bacteria, we cloned recBCD-like genes from diverse bacteria into an E. coli recBCD deletion mutant. Clones from seven species of enteric bacteria conferred to this deletion mutant recombination proficiency, Chi hotspot activity in lambda Red- Gam- vegetative crosses, and RecBCD enzyme activities, including Chi-dependent DNA strand cleavage. Three clones from Pseudomonas aeruginosa and Ps. putida conferred recombination proficiency and ATP-dependent nuclease activity, but neither Chi hotspot activity nor Chi-dependent DNA cleavage. These results imply that Chi has been conserved as a recombination-promoting signal for RecBCD-like enzymes in enteric bacteria but not in more distantly related bacteria such as Pseudomonas spp. We discuss the possibility that other, presently unknown, nucleotide sequences serve the same function as Chi in Pseudomonas spp.

Adenosine Triphosphate↗
PubMed1990

Action of RecBCD enzyme on cruciform DNA.

We tested the hypothesis that RecBCD enzyme of Escherichia coli resolves pre-existing Holliday recombination intermediates by examining the action of the purified enzyme on an open-ended DNA cruciform with limited ability to branch migrate. The enzyme cleaved two strands of the cruciform near its base to produce "recombinant" products, with a marked bias in the direction of cleavage. The two nicks necessary to cleave the cruciform were made separately. Cruciforms whose four termini were blocked by synthetic hairpin-shaped oligonucleotides were not detectably nicked by the enzyme. With one terminus open the enzyme made a nick at the base of the cruciform but not a double-strand cut. With two or more termini open the enzyme made double-strand cuts. We infer that RecBCD enzyme molecules must enter the termini of duplex DNA and approach the cruciform from more than one direction in order to cleave it into recombinant products. Previous results on RecBCD-mediated recombination between phage lambda and lambda dv imply that intracellular RecBCD enzyme can approach pre-existing Holliday junctions from only one direction. We infer that intracellular RecBCD enzyme cannot cleave pre-existing Holliday junctions into recombinants and suggest that the enzyme may cleave Holliday junctions in whose formation it participates.

Base Sequence↗
PubMedDate not supplied

RecBC enzyme activity is required for far-UV induced respiration shutoff in Escherichia coli K12.

Shutoff of respiration is one of a number of recA+ lexA+ dependent (SOS) responses caused by far ultraviolet (245 nm) radiation (UV) damage of DNA in Escherichia coli cells. Thus far no rec/lex response has been shown to require the recB recC gene product, the RecBC enzyme. We report in this paper that UV-induced respiration shutoff did not occur in either of these radiation-sensitive derivatives of K12 strain AB1157 nor in the recB recC double mutant. The sbcB gene product is exonuclease I and it has been reported that the triple mutant strain recB recC sbcB has near normal recombination efficiency and resistance to UV. The sbcB strain shut off its respiration after UV but the triple mutant did not show UV-induced respiration shutoff; the shutoff and death responses were uncoupled. We concluded that respiration shutoff requires RecBC enzyme activity. The RecBC enzyme has ATP-dependent double-strand exonuclease activity, helicase activity and several other activities. We tested a recBC+ (double dagger) mutant strain (recC 1010) that had normal recombination efficiency and resistance to UV but which possessed no ATP-dependent double-strand exonuclease activity. This strain did not shut off its respiration. The presence or absence of other RecBC enzyme activities in this mutant is not known. These results support the hypothesis that ATP-dependent double-strand exonuclease activity is necessary for UV-induced respiration shutoff.

Escherichia coli↗
PubMed1986

Lack of UV-induced respiration shutoff in a recF strain of Escherichia coli: temperature conditional suppression at 30 degrees C by the sfrA mutation.

A mutation in the recF gene of Escherichia coli results in a radiation-sensitive strain. The RecF pathway and the RecBC pathway account for nearly all of the conjugative recombination occurring in E. coli. recBC cells are radiation-sensitive and carry only out a small amount of recombination but these deficiencies are suppressed by an sbcB as recombination is shunted to the RecF pathway. A recBC sbcB recF strain is very radiation-sensitive and is devoid of recombination ability. These deficiencies are suppressed by the srfA mutation; srfA is a recA allele. UV-induced respiration shutoff is a recA+, lexA+ and recBC+ dependent. We report in this paper that respiration does not shutoff in a recF strain at 37 and 30 degrees C. an srfA mutation suppresses this lack of respiration shutoff effect in a recF srfA mutant at 30 degrees C but not at 37 degrees C; no suppression by this mutation occurs at either temperature in a recF recBC sbcB strain. An srfA strain also does not shut off its respiration at 37 degrees C and shows a temperature conditional UV-induced respiration shutoff response at 30 degrees C. The srfA mutation is thought to cause an altered RecA protein to be produced and we suggest that at 37 degrees This altered protein is temperature sensitive. We conclude from the results in this paper that the recF gene product is required for UV-induced respiration shutoff and that the RecA protein plays a special role in the induction process.

Bacterial Proteins↗