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PubMed1995

Interspecies gene exchange in bacteria: the role of SOS and mismatch repair systems in evolution of species.

Analysis of interspecies matings between S. typhimurium and E. coli indicates that the genetic barrier that separates these (and perhaps many other) related species is primarily recombinational. The structural component of this barrier is genomic sequence divergence. The mismatch repair enzymes act as potent inhibitors of interspecies recombination, whereas the SOS system acts as an inducible positive regulator. Interspecies mating triggers a RecBC-dependent SOS response in female bacteria that increases recombination mainly through overproduction of the RecA protein. Mismatch repair acts to reduce the mutation rate and recombination between similar sequences, whereas SOS acts to increase both. These opposing activities allow mismatch repair and SOS systems to determine both the rate of accumulation of sequence divergence and the extent of genetic isolation, which are the key components of the speciation process.

Biological Evolution↗
PubMed1984

Stabilization of the cloning vector pACYC184 by insertion of F plasmid leading region sequences.

The leading region of the F plasmid is, by definition, the first part of the plasmid DNA to be transferred to the recipient cell during conjugation. Restriction fragments of the leading region, when cloned into the plasmid vector pACYC184, extended the maintenance of the normally unstable p15A-derived vector replicon in rec+ Escherichia coli K-12 cells. Mutations in the host's general recombination systems were found to influence the maintenance of these hybrid plasmids.

Base Sequence↗
PubMed1992

RecP, a new minor pathway of general recombination in Escherichia coli encoded by plasmid R1drd-19.

Plasmid R1drd-19 markedly improves the recombination deficiency of recB and recBrecC mutants of Escherichia coli K12 as measured by Hfr crosses and increases their resistance to uv inactivation. The effect correlates with the production of an ATP-dependent ds DNA exonuclease in recB/R1drd-19 cells. This paper further investigates the suppressive effect of plasmid R1drd-19 on the recB mutation of E. coli. The gene(s) responsible for the effect was localized to the 13.1-kb EcoRI-C fragment of the resistance transfer factor (RTF) portion of R1drd-19. The plasmid-encoded activity does not merely replace the RecBCD enzyme failure but differs in several significant ways. It promotes a hyper-recombinogenic phenotype, as judged by the phenomenon of super oligomerization of the tester pACYC184 plasmid in recB/R1drd-19 cells and two inter- and intramolecular plasmid recombination test systems. It is probably not inhibited by lambda Gam protein and does not restrict plating of T4gp2 mutant. No significant homology between the E. coli chromosomal fragment carrying recBrecCrecD genes and the EcoRI-C fragment of R1drd-19 was observed. It is suggested that the plasmid-encoded recombination activity is involved in a new minor recombination pathway (designated RecP, for Plasmid). RecP resembles in some traits the RecBCD-independent pathways RecE and RecF but differs in activity and perhaps substrate specificity from the main RecBCD pathway.

Blotting, Southern↗
PubMed1989

The recB gene product is essential for exonuclease V-dependent DNA degradation in vivo.

The recB21 mutation abolishes the exonuclease activity of the RecBCD enzyme (exonuclease V) of Escherichia coli. This might be due to the polar effect of recB21 on expression of the recD gene, the product of which is an essential component of the RecBCD enzyme. To achieve synthesis of the recD gene product, the recD+ plasmid was introduced into the recB21 mutant. Degradation of the endogenous DNA damaged by gamma-rays and degradation of the DNA of a phage T4 gene 2 mutant were nevertheless abnormally small in this strain. Thus, the functional recB gene product is required for the degradative function of the RecBCD enzyme.

DNA Repair↗
PubMed1992

Overproduction of the RecD polypeptide sensitizes Escherichia coli cells to gamma-radiation.

We investigated DNA metabolism in Escherichia coli cells carrying the multicopy recD+ plasmid (pKI13). In the presence of pKI13, the cellular level of the recD gene product (RecD polypeptide) is amplified at least 60-fold. Overproduction of the RecD polypeptide has no effect on UV repair and conjugational recombination. In contrast, high cellular levels of this polypeptide sensitize wild-type cells to gamma-radiation; also, they increase the rate of radiation-induced DNA degradation. A possible mechanism for the enhancement of gamma-ray-induced killing by large amounts of the RecD polypeptide is discussed.

DNA Repair↗
PubMed1988

Nucleotide excision by E. coli DNA polymerase I in proofreading and non-proofreading modes.

Escherichia coli DNA polymerase I exists in at least two distinct kinetic forms. When it binds to a template, the proofreading activity is usually switched off. As the enzyme progresses along the template, it becomes more and more competent for excision. This phenomenon introduces a link between fidelity and processivity. Processivity is best studied when the chain-length distributions of synthesized polymers are stationary. Even then, however, one cannot avoid multiple initiations on a given template by the same molecule of the enzyme. When synthesis is initiated with primers of lengths 15 or 20, a strange phenomenon is observed. It seems that the polymerase starts by hydrolyzing the primer down to a length of 7-10 nucleotides and only then starts to add nucleotides. It does so in a low-accuracy mode, suggesting that, while the exonuclease is clearly active, it does not contribute to proofreading. The warm-up of the proofreading function is therefore reinterpreted as a switch between two modes of behaviour: a mode 1 of low accuracy in which the 3'----5' exonuclease, while active, is uncoupled from the polymerase and does not contribute to proofreading, and a mode 2 of high accuracy in which the exonuclease is kinetically linked to the polymerase activity.

DNA Polymerase I↗
PubMed1988

Protein-protein interactions of yeast DNA polymerase III with mammalian and yeast proliferating cell nuclear antigen (PCNA)/cyclin.

We have previously reported the purification of yeast analogs to mammalian DNA polymerase delta and proliferating-cell nuclear antigen (PCNA)/cyclin: DNA polymerase III and yeast PCNA, respectively. Through the use of gel-filtration chromatography, we have studied the interaction of the model template-primer system poly(dA).(dT)16 (40:1) with yeast DNA polymerase III and with PCNAs. Yeast DNA polymerase III binds to the DNA in the absence of yeast PCNA/cyclin, but comigration of either yeast or calf thymus PCNA/cyclin with the DNA requires the additional presence of yeast DNA polymerase III. We could also isolate a DNA-calf thymus DNA polymerase delta-calf thymus PCNA/cyclin complex. From these data, we propose that PCNA/cyclin is involved not in the binding step of the polymerase to the template-primer, but in the elongation step. The 3'----5' exonuclease associated with yeast DNA polymerase III acts in a distributive manner on poly(dA).(pT)16, and dissociates from the DNA when addition of dTTP allows switching from the exonuclease to the polymerase mode. Addition of PCNA/cyclin had no effect on these activities.

Animals↗
PubMed1988

The herpes simplex virus DNA polymerase: analysis of the functional domains.

The structural and functional organization of the herpes simplex virus type I (HSV-1) DNA polymerase enzyme of strain ANG was studied by a combination of sequence and immunobiochemical analyses. Comparison of the HSV-1 ANG DNA polymerase sequence with those of pro- and eukaryotic DNA polymerases resulted in the allocation of eleven conserved regions within the HSV-1 DNA polymerase. From the analysis of all currently identified mutations of temperature-sensitive and drug-resistant HSV-1 DNA polymerase mutants as well as from the degree of conservancy observed, it could be deduced that the amino-acid residues 597-961, comprising the homologous sequence regions IV-IX, constitute the major structural components of the catalytic domain of the enzyme which should accommodate the sites for polymerizing and 3'-to-5' exonucleolytic functions. Further insight into the structural organization was gained by the use of polyclonal antibodies responding specifically to the N-terminal, central and C-terminal polypeptide domains of the ANG polymerase. Each of the antisera was able to immunostain as well as to immunoprecipitate a viral polypeptide of 132 +/- 5 kDa that corresponded well to the molecular mass of 136 kDa predicted from the coding sequences. Enzyme-binding and neutralization studies confirmed that both functions, polymerase and 3'-to-5' exonuclease, are intimately related to each other, and revealed that, in addition to the sequences of the proposed catalytic domain, the very C-terminal sequences, except for amino-acid residues 1072-1146, are important for the catalytic functions of the enzyme, most likely effecting the binding to DNA.

Amino Acid Sequence↗
PubMed1988

Partial purification and characterization of a cellular protein that binds to the SV40 core origin of DNA replication.

A monkey cell factor that interacts specifically with double- and single-stranded DNA sequences in the early domain of the simian virus 40 (SV40) core origin of replication was identified using gel-retention assays. The protein was enriched over 1200-fold using ion-exchange and affinity chromatography on single-strand DNA cellulose. Binding of protein to mutant origin DNA restriction fragments was correlated with replication activity of the mutant DNAs. Exonuclease footprint experiments on single-stranded DNA revealed prominent pause sites in the early domain of the core origin. The results suggest that this cellular protein may be involved in SV40 DNA replication.

Base Sequence↗
PubMed1988

Protein-primed replication of bacteriophage phi 29 DNA.

The replication of phi 29 DNA-protein p3 represents a simple model system to study the protein-priming mechanism of initiation of replication. The phi 29 DNA polymerase involved both in the initiation and elongation steps of phi 29 DNA-protein p3 replication, is a very processive enzyme and it is able to produce strand-displacement in the absence of other proteins. To correlate functional and structural domains in the phi 29 DNA polymerase point mutants in the most carboxyl region of amino-acid homology with other DNA polymerases have been constructed. Most of the mutations had a decreased initiation and elongation activity, but normal 3'----5' exonuclease activity, suggesting that this region contributes to the active domain for initiation and elongation. Point and deletion mutants in the terminal protein have allowed the mapping of one DNA-binding region and two DNA-polymerase-binding regions. The viral protein p6, which stimulates the initiation of replication, binds to a set of specific signals present at both phi 29 DNA ends. A good correlation of binding and stimulation of replication has been obtained by using fragments containing phi 29 DNA-terminal sequences and deletion mutants of protein p6. The viral protein p5 has been shown to bind to single-stranded DNA, to protect the latter against nuclease digetion, and to stimulate phi 29 DNA-protein p3 replication in vitro.

Bacteriophages↗
PubMed1995

Inactivation of the 5'-3' exonuclease of Thermus aquaticus DNA polymerase.

The gene for Thermus aquaticus (Taq) DNA polymerase enzyme (Taq Pol I) was mutagenized and sixty-two candidate clones were screened for enzyme activity. Two of the clones expressed enzymes (*Taq-3 and *Taq-5) that showed very reduced 5'-3' exonuclease activity and normal DNA polymerase activity. These two enzymes showed heat resitance and storage stability similar to Taq Pol I and had similar effectiveness in PCR. Processivity of the polymerases was compared by measuring the extension of an end-labeled primer annealed to a single stranded DNA, as well as by a PCR method. The processivity of *Taq-3 and *Taq-5 was similar to Taq Pol I (50-80 nucleotides) and more processive than a Taq Pol I deficient in the 5'-3' exonuclease due to absence of the first 290 amino acids (Stoffel fragment). The results indicate two amino acids which are required for normal 5'-3' exonuclease activity in Taq Pol I (Arg-25 and Arg-74).

Cloning, Molecular↗
PubMed1996

Construction of single amino acid substitution mutants of cloned Bacillus stearothermophilus DNA polymerase I which lack 5'-->3' exonuclease activity.

Two individual amino acid substitutions were engineered at a selected site in the 5' --> 3' exonuclease domain of the cloned Bacillus stearothermophilus DNA polymerase I gene. These mutations resulted in the expression of enzymes lacking the 5' --> 3' exonuclease activity while maintaining normal polymerizing activity. The mutated and non-mutated enzymes were each constitutively expressed in an Escherichia coli host without the use of an exogenous or inducible promoter, and the mutated enzymes were demonstrated to be equivalent to the subtilisin large fragment of the native holoenzyme in sequencing reactions.

Amino Acids↗
PubMed1985

Uvr-independent repair of 8-methoxypsoralen crosslinks in Escherichia coli: evidence for a recombinational process.

On the basis of survival data, repair of 8-methoxypsoralen DNA crosslinks in Escherichia coli strains lacking a functional uvrABC endonuclease, is shown to require the products of the recA, recB, recF and recN genes. Bacteria, grown under conditions where most cells contain only a single genome, show no evidence of crosslink repair. Similarly, bacteriophage lambda shows evidence of crosslink repair only in SOS-induced cells, and only at multiplicities of infection greater than 1. The requirement for rec+ genes may be partly ascribed to the need for a functional SOS response, but taken together, the results suggest a recombinational step involving a homologous region of DNA may occur during uvr-independent crosslink repair.

Bacterial Proteins↗
PubMed1985

A minor pathway of postreplication repair in Escherichia coli is independent of the recB, recC and recF genes.

After ultraviolet (UV) irradiation, an Escherichia coli K12 uvrB5 recB21 recF143 strain (SR1203) was able to perform a limited amount of postreplication repair when incubated in minimal growth medium (MM), but not if incubated in a rich growth medium. Similarly, this strain showed a higher survival after UV irradiation if plated on MM versus rich growth medium (i.e., it showed minimal medium recovery (MMR]. In fact, its survival after UV irradiation on rich growth medium was similar to that of a uvrB5 recA56 strain, which does not show MMR or postreplication repair. The results obtained with a uvrB5 recF332::Tn3 delta recBC strain and a uvrB5 recF332::Tn3 recB21 recC22 strain were similar to those obtained for strain SR1203, suggesting that the recB21 and recF143 alleles are not leaky in strain SR1203. The treatment of UV-irradiated uvrB5 recB21 recF143 and uvrB5 recF332::Tn3 delta recBC cells with rifampicin for 2 h had no effect on survival or the repair of DNA daughter-strand gaps. Therefore, a pathway of postreplication repair has been demonstrated that is constitutive in nature, is inhibited by postirradiation incubation in rich growth medium, and does not require the recB, recC and recF gene products, which control the major pathways of postreplication repair.

Bacterial Proteins↗
PubMed1986

5-Azacytidine: survival and induction of the SOS response in Escherichia coli K-12.

Survival and induction of the SOS system by 5-azacytidine, an analog of cytidine, were studied in Escherichia coli K-12. This compound did not produce any effect on the viability of dcm and dam dcm mutants. Furthermore, recA430 and lexA1 strains (both mutations interfere with LexA repressor cleavage but not recombination proficiency) were more resistant than the wild-type strain of E. coli K-12. In contrast, recBC and recA13 mutants were more sensitive to 5-azacytidine than the wild type. Transient exposure of E. coli to 5-azacytidine for 60 min induced both recA-dependent inhibition of cell division and induction of lambda prophage in Dcm+ strains but not in Dcm- mutants. Expression of both functions was dependent on recBC exonuclease. On the other hand, 5-azacytidine was unable to trigger the induction of umuCD and mucB genes and no amplification of RecA protein synthesis in either Dcm+ or Dcm- strains was observed. These last results are in agreement with previously reported data suggesting that there is a discrimination in the expression of the several SOS functions and that some SOS genes may be induced without amplification of RecA protein synthesis.

Adenosine Triphosphate↗
PubMed1986

Role of the radB gene in postreplication repair in UV-irradiated Escherichia coli uvrB.

In UV-irradiated Escherichia coli, the radB101 mutation sensitized uvrB recF cells 4-fold and uvrB recB cells 1.2-fold, but did not sensitize uvrB recB recF cells. The radB mutation had very little effect (1.2-fold or less) on the repair of UV radiation-induced DNA daughter-strand gaps in uvrB cells, but it did cause about a 3-fold deficiency in the repair of the DNA double-strand breaks that arise in association with nonrepaired daughter-strand gaps in UV-irradiated uvrB recF cells. Thus, the radB gene does not appear to be involved in the recF-dependent or recF recB-independent processes for the repair of DNA daughter-strand gaps, but is involved in the recB-dependent postreplication repair of DNA double-strand breaks.

Bacterial Proteins↗
PubMed1986

Repair of DNA double-strand breaks in UV-irradiated Escherichia coli uvrB recF cells is inhibited by rich growth medium.

Ultraviolet (UV)-irradiated uvrB recF and uvrB recB cells of Escherichia coli K-12 showed similar radiation sensitivities when plated on minimal growth medium (MM), however, the uvrB recF cells were much more UV radiation-sensitive than the uvrB recB cells when plated on rich growth medium. Sedimentation analysis of the DNA from UV-irradiated uvrB recF cells suggests that the rich medium killing of uvrB recF cells is due to the inhibition of the repair of UV-radiation-induced DNA double-strand breaks, i.e., the killing is due to the inhibition of the recB-dependent pathway of postreplication repair. Furthermore, we demonstrated that the DNA double-strand breaks that were formed in UV-irradiated uvrB recA200(Ts) cells incubated at 42 degrees C in rich growth medium were not repaired whether the medium during subsequent repair incubation at 30 degrees C was MM or rich growth medium, while DNA double-strand breaks that were formed in MM at 42 degrees C could be repaired in MM or in rich growth medium at 30 degrees C. How the absence of an abrupt slowing of DNA synthesis when UV-irradiated cells are held in rich growth medium (Sharma and Smith, 1985b) may prevent the repair of these DNA double-strand breaks is discussed.

Culture Media↗
PubMed1986

The influence of repair systems on the presence of sensitive and resistant fractions in the relation of survival of colony-forming ability in Escherichia coli to UV exposure.

In the experimentally observed relationship between survival of colony-forming ability and the amount of exposure to ultraviolet light, two characteristics are generally found. First, sensitive and resistant components often show. Second, there is often a shouldered character to the survival. We present evidence that the first is largely due to the presence of active replication forks in the genome, and that the second is related to the operation of the recombinational repair system. We are able to describe our data in terms of a superposition of single and multiple-hit fractions and to show that the latter are greatly increased, in excision-repair-competent strains, by prevention of protein synthesis for 1 h prior to irradiation. Applying this analysis and treatment to a number of mutant strains enables us to make suggestions as to the interaction between recombinational and excision repair.

DNA Repair↗