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)

We describe the polypeptide structure and some of the catalytic properties of a DNA polymerase alpha.DNA primase complex that can be prepared from KB cells by immunoaffinity purification. The procedure is based on monoclonal antibodies that were raised against a biochemically purified, catalytically active core protomer of the polymerase. In all respects tested, the basic mechanism of substrate recognition and binding by the immunoaffinity-purified polymerase is qualitatively identical to that of the core protomer. The immunoaffinity-purified KB cell polymerase alpha X DNA primase is structurally complex. On the basis of extensive immunochemical analyses with five independent monoclonal antibodies, three of which are potent neutralizers of polymerase alpha activity, peptide mapping studies, and the application of a sensitive immunoassay that permits detection of polymerase alpha antigens in crude cell lysates, we have established that the principal form of catalytically active DNA polymerase alpha in KB cells is a phosphoprotein with a molecular mass of 180 kilodaltons. This protein is stable in vivo, with an estimated half-life of greater than or equal to 15 h. In contrast, the polypeptide is extremely fragile in vitro and generates partial degradation products of p165, p140, and p125 that explain the "microheterogeneity" typically exhibited by polymerase alpha peptides in denaturing polyacrylamide gels. In addition to the catalytically active polymerase alpha polypeptide(s), the immunopurified enzyme fraction typically contains three other proteins, p77, p55, and p49, the functions of which have not yet been established. These proteins do not display polymerase alpha epitopes and have been shown by peptide mapping to be independent species that are unrelated either to the large polymerase peptides or to one another. The polypeptide p77 is also a phosphoprotein, and in both p180 and p77 the phosphorylated amino acids are exclusively serine and threonine.
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PMID:Structural and enzymological characterization of immunoaffinity-purified DNA polymerase alpha.DNA primase complex from KB cells. 242 27

The Bacillus subtilis dnaF (polC) gene that codes for the alpha subunit of the DNA polymerase III holoenzyme has been sequenced. It consists of 4005 base pairs coding for 1335 amino acids (from the start to the stop codon), giving a molecular weight of 151,273. A mutation (azp-12) that confers resistance to the antimicrobial drug 6-(p-hydroxyphenylazo)-uracil is due to a single base change at nucleotide 3523, from TCA to GCA, resulting in a change of the 1175th amino acid, serine, to alanine. It is in the active site and located at the C-terminal part of the enzyme. The amino acid composition in an N-terminal domain has 26% homology to the epsilon subunit coded by the dnaQ gene of Escherichia coli, which is a 3'----5' proofreading exonuclease, supporting an earlier observation that this function is an integral part of the polymerase molecule in B. subtilis.
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PMID:DNA polymerase III gene of Bacillus subtilis. 249 83

Wild type (wt) Bacillus subtilis polC and polCazp12, a mutant derivative specifying a form of DNA polymerase III resistant to hydroxyphenylazopyrimidines, were cloned as genomic fragments approximating the length required to encode the entire polymerase. The cloned DNA fragments were subjected to restriction and partial sequence analysis to locate the 5' end of the polC-specific coding sequence and the azp12 mutation, which was identified as a T----G transversion specifying replacement of serine with alanine. The cloned wt and azp12-coding sequences were recloned in an Escherichia coli expression vector with their respective 5' ends under the control of the bacteriophage lambda PL promoter and cIts857-encoded repressor. In response to induction, the wt- and azp12-specific recombinant plasmids expressed active DNA polymerases indistinguishable from the native enzymes derived from the respective B. subtilis hosts.
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PMID:The cloned polC gene of Bacillus subtilis: characterization of the azp12 mutation and controlled in vitro synthesis of active DNA polymerase III. 251 95

A mutation (asparagine 815 to serine 815) was introduced into the herpes simplex virus type 1 (HSV-1) DNA polymerase (pol). The HSV-1 pol enzyme in lysates of Saccharomyces cerevisiae cells expressing the mutant protein showed increased resistance to acyclovir triphosphate and increased sensitivity to phosphonoacetate but was not substantially altered with respect to sensitivity to phosphonoformate or aphidicolin. These results directly demonstrate that both resistance to acyclovir triphosphate and sensitivity to phosphonoacetate can be conferred by this mutation in the absence of other viral factors and that the yeast expression system can be used for structure-function studies on HSV-1 pol.
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PMID:In vitro mutagenesis of the herpes simplex virus type 1 DNA polymerase gene results in altered drug sensitivity of the enzyme. 255 70

The amino acid sequence, arginine-glycine-aspartic acid (RGD), found in some cell adhesive proteins, is a recognition signal for the receptor protein. It is interesting that we have found the RGD sequence in terminal protein (TP) of bacteriophages phi 29 and M2 near an amino acid, the serine residue at 232, covalently linked to the terminal nucleotide of their DNAs. At the initiation of protein-primed DNA replication, TP is essential for the recognition of replication machinery containing DNA polymerase and primer protein (PP; PP becomes TP upon linking the first nucleotide, and hence the primary structure of TP is the same as that of PP). Synthetic peptide RGD specifically inhibited transfection of phi 29 and M2. The target of the RGD peptide is shown to be TP by marker rescue experiments, suggesting that a receptor for the RGD sequence exists in TP. Furthermore, the peptide inhibited the in vitro protein-priming reaction of DNA replication. We propose that the RGD sequence of PP and a putative receptor on TP is utilized for the molecular recognition initiating DNA replication.
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PMID:An inhibitory effect of RGD peptide on protein-priming reaction of bacteriophages phi 29 and M2. 260 28

To test the utility of the polymerase chain reaction in identifying single base mutations in a gene known to give rise to an altered enzyme and drug resistance phenotype, a human colon adenocarcinoma cell line resistant to methotrexate, with a known single base mutation (Srimatkandada et al., J. Biol. Chem. 264:3524, 1989) was examined. Poly A+ RNA was used for cDNA synthesis with reverse transcriptase, deoxynucleoside triphosphates, and 5 microM 3' primer that anneals outside the coding region of the human dihydrofolate reductase. The RNA:DNA hybrid was used as a template for the polymerase chain reaction with the addition of a 5' primer and Thermus aquaticus (Taq)I DNA polymerase. These primers flank the coding region of the human dihydrofolate reductase and define a region of 650 bases. The polymerase chain reaction was carried out for 40 cycles resulting in full length transcripts in microgram amounts clearly visible by ethidium bromide staining on agarose gels. DNA was isolated by standard methods, and double-stranded DNA was sequenced by the chain-termination method using TaqI DNA polymerase. A single point mutation was discovered at position 91 (T----C) resulting in a substitution of serine for phenylalanine at codon 31, as determined previously by classical cDNA cloning and sequencing. Sequence analysis indicated that this base transition resulted in the loss of Eco RI and Xmn I sites and the gain of a HinfI site in the cDNA, which were confirmed by restriction digests.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Detection of a single base mutation in the human dihydrofolate reductase gene from a methotrexate-resistant cell line using the polymerase chain reaction. 264 Jan 57

Before an oxic cell sensitizer such as beta-ara A (a DNA-dependent DNA polymerase inhibitor) can be used in cancer treatment, it is essential to know both the influence of this type of drug on certain critical normal tissues and the role of proliferation kinetics in the radiosensitizing capacity. The biological system chosen for this in vitro study was the human fibroblast cell line HF19. Cells were studied in plateau phase and in the exponential growth phase. Cells were incubated with beta-ara A for 7 hr (1 hr before and 6 hr after irradiation). beta-ara A was extremely toxic to growing cells (concentrations ranging from 200 to 1000 microM), but no detectable effect was found on plateau-phase cells (up to 4000 microM). However, for a given drug concentration, the radiosensitizing effect (Sensitizing Enhancement Ratio SER) was very similar for growing and plateau phase cells (SER measured with Ds ratio was about 1.7 for a concentration of 500 microM). The enhancement ratio depended on the radiation dose; it was relatively higher for low doses. This can be explained by a differential effect of the drug on the alpha and beta components of the survival curve. Only the alpha component was increased.
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PMID:The effect of the oxic cell sensitizer, beta-ara A on human fibroblasts in plateau and in exponential growth phases. 271 77

Mechanism-based enzyme inactivator, alanine racemase, S-adenosylhomocysteine hydrolase, D-amino acid aminotransferase, gamma-aminobutyric acid aminotransferase, arginine decarboxylase, aromatase, L-aromatic amino acid decarboxylase, dihydrofolate reductase, dihydroorotate dehydrogenase DNA polymerase I, dopamine beta-hydroxylase, histidine decarboxylase, beta-lactamase, monoamine oxidase, ornithine decarboxylase, serine proteases, testosterone 5 alpha-reductase, thymidylate synthetase, xanthine oxidase.
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PMID:The potential use of mechanism-based enzyme inactivators in medicine. 306 67

By site-directed mutagenesis we have changed the serine residue 232 of the phi 29 terminal protein, involved in the covalent linkage to dAMP for the initiation of replication, into a threonine residue. The mutant terminal protein has been purified to homogeneity and shown to be inactive in the formation of the initiation complex; nevertheless, the mutant protein retains its ability to interact with the phi 29 DNA polymerase and with the DNA. The results obtained indicate a high specificity in the linking site of the terminal protein.
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PMID:Site-directed mutagenesis in the DNA linking site of bacteriophage phi 29 terminal protein: isolation and characterization of a Ser232----Thr mutant. 313 31

We describe a procedure by which the codon (AGC) for the active-site serine-70 of pBR322 beta-lactamase (penicillinase, penicillin amido-beta-lactamhydrolase, EC 3.5.2.6) is altered to that for cysteine (TGC). The pertinent nucleotide bases, A-G-C-A, positions 410-413, of pBR322 are excised by treating a limited HgiAI digest of pBR322 with the 3' leads to 5' exonuclease of T4 DNA polymerase. The new sequence, T-G-C-A, is inserted in two steps. First, the Kpn I molecular linker d(T-G-G-T-A-C-C-A) is ligated into the gap described above. The internal sequence G-T-A-C is then excised enzymatically with Kpn I and T4 DNA polymerase and the molecule is recircularized. This mutant gene, which codes for a thiol-beta-lactamase, confers on Escherichia coli K-12 hosts an ampicillin resistance that is reduced compared with that given by pBR322 yet is greater than that of E. coli lacking any intact beta-lactamase gene. Cell-free extracts of E. coli strains hosting the thiol-beta-lactamase gene possess a p-chloromercuribenzoate-sensitive beta-lactamase activity.
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PMID:Thiol-beta-lactamase: replacement of the active-site serine of RTEM beta-lactamase by a cysteine residue. 681 41


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