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:1.5.1.3 (dihydrofolate reductase)
5,819 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

We have used three methods to study the formation and repair of intrastrand adducts and interstrand cross-links in the DNA of Chinese hamster ovary cells induced by the anticancer drug cis-diamminedichloroplatinum II (cisplatin). Using atomic absorption spectroscopy, we found that 21% of the total genomic cisplatin adducts were removed at 8 h and 42% at 24 h. We used ABC excinuclease digestion, coupled with out previously reported methodology to quantify DNA in specific genomic regions. These adducts were removed faster in the transcribed dihydrofolate reductase and c-myc genes compared to a noncoding fragment, a region containing the little or nontranscribed c-fos oncogene, and to the overall genome. Interstrand cross-links in specific sequences were quantified by Southern hybridization of denatured-renatured DNA separated on a neutral gel. We found that cross-links were removed more efficiently from the gene regions than intrastrand adducts and, at high levels of cross-linking, removal was similar from transcribed and from nontranscribed regions.
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PMID:Gene-specific formation and repair of cisplatin intrastrand adducts and interstrand cross-links in Chinese hamster ovary cells. 201 18

We have previously demonstrated preferential DNA repair of active genes in mammalian cells. The methodology involves the use of a specific endonuclease or other more direct approaches to create nicks at sites of damage followed by quantitative Southern analysis and probing for specific genes. Initially, we used pyrimidine dimer specific endonuclease to detect pyrimidine dimers after UV irradiation. We now also use the bacterial enzyme ABC excinuclease to examine the DNA damage and repair of a number of adducts other than pyrimidine dimers in specific genes. We can detect gene specific alkylation damage by creating nicks via depurination and alkaline hydrolysis. In our assay for preferential repair, we compare the efficiency of repair in the DHFR gene to that in the 3' flanking, non-coding region to the gene. In CHO cells, UV induced pyrimidine dimers are efficiently repaired from the active DHFR gene, but not from the inactive region. We have demonstrated that the 6-4 photoproducts are also preferentially repaired and that they are removed faster from the regions studied than pyrimidine dimers. Using similar approaches, we find that DNA adducts and crosslinks caused by cisplatinum are preferentially repaired in the active gene compared to the inactive regions and to the inactive c-fos oncogene. Also, nitrogen mustard and methylnitrosurea damage is preferentially repaired whereas dimethylsulphate damage is not. NAAAF adducts do not appear to be preferentially repaired in this system.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Gene specific damage and repair after treatment of cells with UV and chemotherapeutical agents. 206 87

DNA repair in man can be described in general terms, but details are still obscure. Excision repair of base damage has a general similarity to the mechanism of the bacterial uvr ABC exonuclease, but the individual roles of at least 15 genes that regulate mammalian excision repair are as yet unknown. The differential repair of specific regions of DNA and of specific genes is highlighted by the clustered mode of repair characteristic of xeroderma pigmentosum group C and by the rapid repair of the dihydrofolate reductase gene. Cloning of genes that specify repair in man is proceeding slowly, in part, because of confusion by genes that produce only partial correction or nonspecific changes in sensitivity and by phenotypic reversion. In human cells, DNA damage-inducible genes are recognized that may overlap the spectra of other stress-induced proteins, but the relationship of these to any error-prone or recA-like system is unknown and unlikely. Four diseases, xeroderma pigmentosum, ataxia telangiectasia, Cockayne syndrome, and Fanconi anemia, have well-documented and significantly increased sensitivities to DNA-damaging agents, and each has recognizable though complex abnormalities in processing DNA damage. In addition, a wide variety of diseases and cellular processes have been ascribed to an association with DNA damage and repair, but the accuracy and significance of these associations are hard to identify.
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PMID:DNA repair in man. 265 41

We have developed a method to quantify (6-4) photoproducts in genes and other specific sequences within the genome. This approach utilizes the following two enzymes from Escherichia coli: ABC excinuclease, a versatile DNA repair enzyme which recognizes many types of lesions in DNA, and DNA photolyase, which reverts pyrimidine dimers. DNA is isolated from UV irradiated Chinese hamster ovary cells and digested with a restriction enzyme. Pyrimidine dimers, the major photoproduct produced at biological UV fluences, are then completely repaired by treatment with DNA photolyase. The photoreactivated DNA is treated with ABC excinuclease, electrophoresed in an alkaline agarose gel, transferred to a support membrane and probed for specific genomic sequences. Net incisions produced by ABC excinuclease following photoreactivation are largely due to the presence of (6-4) photoproducts. These adducts are quantitated by measuring the reduction of intensity of the full length fragments on the autoradiogram. Using this approach we have shown that (6-4) photoproducts are produced at equal frequency in the dihydrofolate reductase coding sequence and in its 3'-flanking, noncoding sequences and that the formation of (6-4) photoproducts is linear in both sequences up to a UV dose of 60 J/m2. The repair of (6-4) photoproducts in these DNA sequences was measured after a dose of 40 J/m2 over 4-, 8-, and 24-h time periods. The (6-4) photoproducts are repaired more efficiently than pyrimidine dimers in both sequences and there is preferential repair of (6-4) photoproducts in the dihydrofolate reductase gene compared with the downstream, noncoding sequences.
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PMID:Preferential DNA repair of (6-4) photoproducts in the dihydrofolate reductase gene of Chinese hamster ovary cells. 280 61

We have analyzed gene-specific and strand-specific DNA damage and repair in the dihydrofolate reductase gene in hamster cells. Cells were UV-irradiated or treated with two types of chemotherapeutics, alkylating agents or cisplatin. UV-induced pyrimidine dimers were detected using a previously published technique in which the T4 endonuclease V enzyme is used to create nicks at the lesion sites. 6-4 photoproducts were detected in a similar assay using ABC excinuclease after prior reversal of the pyrimidine dimers with photolyase. Adducts formed by the alkylating agents nitrogen mustard and dimethyl sulfate were quantitated by generating strand breaks at basic sites after neutral depurination. Cisplatin-induced intrastrand adducts were detected with ABC excinuclease, and cisplatin interstrand cross-links were detected using a denaturation-reannealing reaction before electrophoresis. In accord with previous reports by other investigators, we find distinct strand specificity of the repair of pyrimidine dimers after UV; the transcribed strand was much more efficiently repaired than the nontranscribed strand. In contrast, there was little or no strand bias in the repair of the 6-4 photoproducts. For alkylating agents, a slight bias toward repair in the transcribed strand was found after treatment with nitrogen mustard, but there appeared to be no bias in the repair after treatment with dimethyl sulfate. Cisplatin interstrand cross-links are repaired with equal efficiency from the two strands, but the more common cisplatin-induced lesion, the intrastrand adduct, is preferentially repaired from the transcribed strand. In conclusion, there is strand bias in the repair of pyrimidine dimers and cisplatin intrastrand adducts, but the strand specificity of repair may not be a general feature for all DNA lesions, as we found little or no strand bias in the repair of other lesions studied.
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PMID:Repair of individual DNA strands in the hamster dihydrofolate reductase gene after treatment with ultraviolet light, alkylating agents, and cisplatin. 842 Sep 40

A region of 14.2 kb has been analysed that is a part of a locus on the Methylobacterium extorquens AM1 chromosome containing a number of genes involved in one-carbon (C1) metabolism, including serine cycle genes, pqq genes, regulatory methanol oxidation genes and the gene for N5,N10-methylene tetrahydrofolate dehydrogenase (mtdA). Fifteen new ORFs have been identified within the new region, and their sequences suggest that they encode the following polypeptides: the C-terminal part of phosphoenolpyruvate carboxylase, malyl-CoA lyase, polypeptides of 9.4 and 31 kDa of unknown function, three putative subunits of an ABC-type transporter, two polypeptides similar to the products of mxaF and mxaJ from M. extorquens AM1 and other methylotrophs, a cytochrome c, three enzymes of folate metabolism, and polypeptides of 13 and 20.5 kDa with no homologues in the protein database. Ten insertion mutations have been generated in the region to determine if the newly identified genes are associated with C1 metabolism. A mutation in mclA, encoding malyl-CoA lyase, resulted in a C1-minus phenotype, while mutations in the other genes all showed a C1-plus phenotype. It was not possible to obtain null mutants in a putative folate metabolism gene, folC, implying the necessity of these folate synthesis genes for metabolism of C1 and multicarbon compounds. Mutations in the putative ABC transporter genes, the genes similar to mxaG and mxaJ, and other unidentified ORFs produced double-crossover recombinants with a C1-positive phenotype. Promoter regions have been investigated upstream of orf3 and orf4 using the promoter probe vector pHX200. Transcription from these promoters was weak in wild-type M. extorquens AM1 but increased in regulatory mox mutants.
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PMID:Molecular and mutational analysis of a DNA region separating two methylotrophy gene clusters in Methylobacterium extorquens AM1. 916 22

In this study we describe molecular mechanisms of resistance to several classes of antibiotics within drug targets by in silico genome comparisons for bacteria of the genus Rickettsia. Apart from the mutations in the rpoB gene in naturally rifampin-resistant Rickettsia species previously reported by our team, we found that typhus group (TG) rickettsiae had a triple amino acid difference in the highly conserved region of the L22 ribosomal protein as compared to the spotted fever group rickettsiae (SFG), which could explain the natural resistance of SFG rickettsia to erythromycin. We found also that the genome of R. conorii contains an aminoglycoside 3'-phosphotransferase. Finally, either folA gene (encoding dihydrofolate reductase) and/or folP gene (encoding dihydropteroate synthase) was missing in the genome of rickettsial strains explaining the natural resistance to cotrimoxazole. Finally, multiple genes encoding for pump efflux were found especially in the genome of R. conorii that could be involved in resistance to antibiotics. Five specific ORFs related to antibiotic resistance have been identified in the genome of R. felis including a streptomycin resistance protein homologue, a class C beta-lactamase, a class D beta-lactamase, a penicillin acylase homologue, and an ABC-type multidrug transporter system. For the first time, using this approach, an experimental beta-lactamase activity has been shown for this bacterium. We believe that whole genome sequence analysis may help to predict several phenotypic characters, in particular resistance to antibiotics for obligate intracellular bacteria.
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PMID:Genome comparison analysis of molecular mechanisms of resistance to antibiotics in the Rickettsia genus. 1648 18

The prevalence of lung disease due to infections with nontuberculous mycobacteria (NTM) has been increasing and surpassed tuberculosis (TB) in some countries. Treatment outcomes are often unsatisfactory, highlighting an urgent need for new anti-NTM medications. Although NTM in general do not respond well to TB specific drugs, the similarities between NTM and Mycobacterium tuberculosis at the molecular and cell structural level suggest that compound libraries active against TB could be leveraged for NTM drug discovery. Here we tested this hypothesis. The Pathogen Box from the Medicines for Malaria Venture (MMV) is a collection of 400 diverse drug-like compounds, among which 129 are known to be active against M. tuberculosis. By screening this compound collection against two NTM species, Mycobacterium abscessus and Mycobacterium avium, we showed that indeed the hit rates for NTM among TB active compounds is significantly higher compared to compounds that are not active against TB. MIC/dose response confirmation identified 10 top hits. Bactericidal activity determination demonstrated attractive potency for a subset of the confirmed hits. In vivo pharmacokinetic profiling showed that some of the compounds present reasonable starting points for medicinal chemistry programs. Three of the top hits were oxazolidinones, suggesting the potential for repositioning this class of protein synthesis inhibitors to replace linezolid which suffers from low potency. Two hits were inhibitors of the trehalose monomycolate transporter MmpL3, suggesting that this transmembrane protein may be an attractive target for NTM. Other hits are predicted to target a range of functions, including cell division (FtsZ), DNA gyrase (GyrB), dihydrofolate reductase, RNA polymerase and ABC transporters. In conclusion, our study showed that screening TB active compounds for activity against NTM resulted in high hit rates, suggesting that this may be an attractive approach to kick start NTM drug discovery projects. In addition, the work identified a series of novel high value NTM hits with associated candidate targets which can be followed up in hit-to-lead projects for the discovery of new NTM antibiotics.
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PMID:Screening of TB Actives for Activity against Nontuberculous Mycobacteria Delivers High Hit Rates. 2886 Oct 54

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