Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:2.7.7.7 (DNA polymerase)
17,007 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The killer plasmid k1 of Kluyveromyces lactis has terminal inverted repeats of 202 base pairs (bp). The left terminal repeat is contiguous to the transcribed open reading frame, ORF1, which is supposed to code for a DNA polymerase. A 266-bp fragment (called Pk1) containing most of the terminal repeat sequence was isolated and examined for promoter activity. Pk1 was fused, in either original or inversed orientation, with a promoter-less lacZ gene of E coli and a promoter-less G418 resistance gene of Tn903. These fusions were introduced into a pKD1-derived circular vector, and transformed into a lactose-negative (lac4), and a G418-sensitive K lactis host. Lac+ and G418-resistant transformants were obtained with either orientation of Pk1. The promoter activity of Pk1 fragment was independent of the presence or absence of killer plasmids. It is not known whether Pk1 can also function bidirectionally on the natural k1 plasmid. The possible functions of Pk1 for killer plasmid gene expression and plasmid replication are discussed.
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PMID:Promoter activity associated with the left inverted terminal repeat of the killer plasmid k1 from yeast. 166 Jul 26

The role of Escherichia coli DNA polymerase (Pol) II in producing or avoiding mutations was investigated by replacing the chromosomal Pol II gene (polB+) by a gene encoding an exonuclease-deficient mutant Pol II (polBex1). The polBex1 allele increased adaptive mutations on an episome in nondividing cells under lactose selection. The presence of a Pol III antimutator allele (dnaE915) reduced adaptive mutations in both polB+ cells and cells deleted for polB (polB delta 1) to below the wild-type level, suggesting that both Pol II and Pol III are synthesizing episomal DNA in nondividing cells but that in wild-type cells Pol III generates the adaptive mutations. The adaptive mutations were mainly -1 frame-shifts occurring in short homopolymeric runs and were similar in wild-type, polB delta 1, and polBex1 strains. Mutations produced by both Pol III and Pol II ex1 were corrected by the mutHLS mismatch repair system.
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PMID:Proofreading-defective DNA polymerase II increases adaptive mutation in Escherichia coli. 764 19

DNA polymerase II (Pol II) is regulated as part of the SOS response to DNA damage in Escherichia coli. We examined the participation of Pol II in the response to oxidative damage, adaptive mutation, and recombination. Cells lacking Pol II activity (polB delta 1 mutants) exhibited 5- to 10-fold-greater sensitivity to mode 1 killing by H2O2 compared with isogenic polB+ cells. Survival decreased by about 15-fold when polB mutants containing defective superoxide dismutase genes, sodA and sodB, were compared with polB+ sodA sodB mutants. Resistance to peroxide killing was restored following P1 transduction of polB cells to polB+ or by conjugation of polB cells with an F' plasmid carrying a copy of polB+. The rate at which Lac+ mutations arose in Lac- cells subjected to selection for lactose utilization, a phenomenon known as adaptive mutation, was increased threefold in polB backgrounds and returned to wild-type rates when polB cells were transduced to polB+. Following multiple passages of polB cells or prolonged starvation, a progressive loss of sensitivity to killing by peroxide was observed, suggesting that second-site suppressor mutations may be occurring with relatively high frequencies. The presence of suppressor mutations may account for the apparent lack of a mutant phenotype in earlier studies. A well-established polB strain, a dinA Mu d(Apr lac) fusion (GW1010), exhibited wild-type (Pol II+) sensitivity to killing by peroxide, consistent with the accumulation of second-site suppressor mutations. A high titer anti-Pol II polyclonal antibody was used to screen for the presence of Pol II in other bacteria and in the yeast Saccharomyces cerevisiae. Cross-reacting material was found in all gram-negative strains tested but was not detected in gram-positive strains or in S. cerevisiae. Induction of Pol II by nalidixic acid was observed in E. coli K-12, B, and C, in Shigella flexneri, and in Salmonella typhimurium.
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PMID:Involvement of Escherichia coli DNA polymerase II in response to oxidative damage and adaptive mutation. 792 92

In PCR, DNA polymerases from thermophilic bacteria catalyze the extension of primers annealed to templates as well as the structure-specific cleavage of the products of primer extension. Here we show that cleavage by Thermus aquaticus and Thermus thermophilus DNA polymerases can be precise and substantial: it occurs at the base of the stem-loop structure assumed by the single strand products of primer extension using as template a common genetic element, the promoter-operator of the Escherichia coli lactose operon, and may involve up to 30% of the products. The cleavage is independent of primer, template, and triphosphates, is dependent on substrate length and temperature, requires free ends and Mg2+, and is absent in DNA polymerases lacking the 5'-->3' exonuclease, such as the Stoffel fragment and the T7 DNA polymerase. Heterogeneity of the extension products results also from premature detachment of the enzyme approaching the 5' end of the template.
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PMID:Heterogeneity of primer extension products in asymmetric PCR is due both to cleavage by a structure-specific exo/endonuclease activity of DNA polymerases and to premature stops. 861 Jan 8

We describe a method to generate in vivo collections of mutants orders of magnitude larger than previously possible. The method favors accumulation of mutations in the target gene, rather than in the host chromosome. This is achieved by propagating the target gene on a plasmid, in Escherichia coli cells, within the region preferentially replicated by DNA polymerase I (Pol I), which replicates only a minor fraction of the chromosome. Mutagenesis is enhanced by a conjunction of a Pol I variant that has a low replication fidelity and the absence of the mutHLS system that corrects replication errors. The method was tested with two reporter genes, encoding lactose repressor or lipase. The proportion of mutants in the collection was estimated to reach 1% after one cycle of growth and 10% upon prolonged cell cultivation, resulting in collections of 10(12)-10(13) mutants per liter of cell culture. The extended cultivation did not affect growth properties of the cells. We suggest that our method is well suited for generating protein variants too rare to be present in the collections established by methods used previously and for isolating the genes that encode such variants by submitting the cells of the collections to appropriate selection protocols.
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PMID:Efficient gene targeted random mutagenesis in genetically stable Escherichia coli strains. 1105 45

The neo-Darwinists suggested that evolution is constant and gradual, and thus that genetic changes that drive evolution should be too. However, more recent understanding of phenomena called adaptive mutation in microbes indicates that mutation rates can be elevated in response to stress, producing beneficial and other mutations. We review evidence that, in Escherichia coli, two separate mechanisms of stress-induced genetic change occur that revert a lac frameshift allele allowing growth on lactose medium. First, compensatory frameshift ("point") mutations occur by a mechanism that includes DNA double-strand breaks and (we have suggested) their error-prone repair. Point mutation requires induction of the RpoS-dependent general stress response, and the SOS DNA damage response leading to upregulation of the error-prone DNA polymerase DinB (Pol IV), and occurs during a transient limitation of post-replicative mismatch repair activity. A second mechanism, adaptive amplification, entails amplification of the leaky lac allele to 20-50 tandem repeats. These provide sufficient beta-galactosidase activity for growth, thereby apparently deflecting cells from the point mutation pathway. Unlike point mutation, amplification neither occurs in hypermutating cells nor requires SOS or DinB, but like point mutation, amplification requires the RpoS-dependent stress response. Similar processes are being found in other bacterial systems and yeast. Stress-induced genetic changes may underlie much of microbial evolution, pathogenesis and antibiotic resistance, and also cancer formation, progression and drug resistance.
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PMID:Adaptive mutation and amplification in Escherichia coli: two pathways of genome adaptation under stress. 1520 67

"Adaptive mutation" denotes a collection of processes in which cells respond to growth-limiting environments by producing compensatory mutants that grow well, apparently violating fundamental principles of evolution. In a well-studied model, starvation of stationary-phase lac(-)Escherichia coli cells on lactose medium induces Lac(+)revertants at higher frequencies than predicted by usual mutation models. These revertants carry either a compensatory frameshift mutation or a greater than 20-fold amplification of the leaky lac allele. A crucial distinction between alternative hypotheses for the mechanisms of adaptive mutation hinges on whether these amplification and frameshift mutation events are distinct, or whether amplification is a molecular intermediate, producing an intermediate cell type, in colonies on a pathway to frameshift mutation. The latter model allows the evolutionarily conservative idea of increased mutations (per cell) without increased mutation rate (by virtue of extra gene copies per cell), whereas the former requires an increase in mutation rate, potentially accelerating evolution. To resolve these models, we probed early events leading to rare adaptive mutations and report several results that show that amplification is not the precursor to frameshift mutation but rather is an independent adaptive outcome. (i) Using new high-resolution selection methods and stringent analysis of all cells in very young (micro)colonies (500-10,000 cells), we find that most mutant colonies contain no detectable lac-amplified cells, in contrast with previous reports. (ii) Analysis of nascent colonies, as young as the two-cell stage, revealed mutant Lac(+)cells with no lac-amplified cells present. (iii) Stringent colony-fate experiments show that microcolonies of lac-amplified cells grow to form visible colonies of lac-amplified, not mutant, cells. (iv) Mutant cells do not overgrow lac-amplified cells in microcolonies fast enough to mask the lac-amplified cells. (v)lac-amplified cells are not SOS-induced, as was proposed to explain elevated mutation in a sequential model. (vi) Amplification, and not frameshift mutation, requires DNA polymerase I, demonstrating that mutation is separable from amplification, and also illuminating the amplification mechanism. We conclude that amplification and mutation are independent outcomes of adaptive genetic change. We suggest that the availability of alternative pathways for genetic/evolutionary adaptation and clonal expansion under stress may be exploited during processes ranging from the evolution of drug resistance to cancer progression.
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PMID:Adaptive amplification and point mutation are independent mechanisms: evidence for various stress-inducible mutation mechanisms. 1555 Sep 83

Under non-lethal selective conditions, a non-dividing or very slowly dividing microbial population gives rise to mutations that relieve selective pressures. This process is described as adaptive mutation. Salmonella typhimurium strain 5-28 has been used as a system for studying adaptive mutations in the chromosomal regulatory gene purR and its target, the purD operator. When this strain is plated on a minimal lactose medium, no apparent growth of parent lawn is observed, yet the revertant colonies accumulate over a period of time. Analysis of the purR mutational spectra showed that the frequencies of transitions and transversions were not significantly different among the growth-dependent and adaptive mutations. But the frequencies for five kinds of -1 frameshifts were significantly different between the growth-dependent and adaptive types. Among the growth-dependent mutations, most one-base deletions occurred in non-iterated bases and were distributed randomly. Among adaptive mutations, the frequency of one-base deletions in small mononucleotide repeats was higher and mutations were concentrated at three hotspots. One-base deletion in small mononucleotide repeats are generally believed to result from DNA polymerase slippage errors, which are not corrected by DNA repair machinery. We further investigated the role of DNA repair on adaptive mutation. Our results showed that the mismatch repair (MMR) might function less efficiently during adaptive mutation. However, DNA oxidative damage repair seemed no less effective in correcting errors under selective pressures than during non-selective growth.
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PMID:Adaptive mutations in Salmonella typhimurium phenotypic of purR super-repression. 1641 87

Novel deoxyribonucleotide triphosphates bearing maltose or lactose groups were synthesized as substrates for DNA polymerase. The incorporation efficiencies of these modified substrates were investigated in both primer extension reactions and PCR. The stability and conformation of saccharide-modified dsDNAs were assessed by UV absorbance melting experiments and CD analysis. Enzymatic incorporation of saccharide-modified substrates can be used for the efficient production of saccharide-modified DNAs.
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PMID:Construction of saccharide-modified DNAs by DNA polymerase. 1706 95

In some enterobacterial pathogens, but not in Escherichia coli, loss-of-function mutations are a common route to clinically relevant beta-lactam antibiotic resistance. We previously constructed an assay system for studying enterobacterial beta-lactam resistance mutations using the well-developed genetics of E. coli by integrating enterobacterial ampRC genes into the E. coli chromosome. Like the cells of other enterobacteria, E. coli cells acquire beta-lactam resistance by ampD mutation. Here we show that starvation and stress responses provoke ampD beta-lactam resistance mutagenesis. When starved on lactose medium, Lac(-) strains used in mutagenesis studies accumulate ampD beta-lactam resistance mutations independent of Lac reversion. DNA double-strand break repair (DSBR) proteins and the SOS and RpoS stress responses are required for this mutagenesis, in agreement with the results obtained for lac reversion in these cells. Surprisingly, the stress-induced ampD mutations require DinB (DNA polymerase IV) and partially require error-prone DNA polymerase V, unlike lac mutagenesis, which requires only DinB. This assay demonstrates that real-world stressors, such as starvation, can induce clinically relevant resistance mutations. Finally, we used the ampD system to observe the true forward-mutation sequence spectrum of DSBR-associated stress-induced mutagenesis, for which previously only frameshift reversions were studied. We found that base substitutions outnumber frameshift mutations, as seen in other experimental systems showing stress-induced mutagenesis. The important evolutionary implication is that not only loss-of-function mutations but also change-of-function mutations can be generated by this mechanism.
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PMID:Stress-induced beta-lactam antibiotic resistance mutation and sequences of stationary-phase mutations in the Escherichia coli chromosome. 1964 47


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