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)

(S)-1-[3-Hydroxy-2-(phosphonylmethoxy)propyl]cytosine (HPMPC) is an antiviral phosphonate nucleotide analogue that displays activity against a range of herpesviruses. Anion exchange high performance liquid chromatography analysis of the 60% methanol extract from [14C]HPMPC-treated cells reveals the formation of three major metabolites. Two of these were identified as phosphorylated forms of HPMPC, HPMPC phosphate, and HPMPC diphosphate, by liberation of HPMPC upon acid digestion and coelution with synthetic standards on high performance liquid chromatography. The third metabolite, which is resistant to alkaline phosphatase cleavage but sensitive to phosphodiesterase, is proposed to be an HPMPC phosphate adduct. In herpes simplex virus-1-infected cells the same three metabolites are detected, at concentrations comparable to those in uninfected cells. When HPMPC is removed from the medium, the concentrations of the metabolites in cells decrease slowly, with half-lives of approximately 6, 17, and 48 hr for HPMPC phosphate, HPMPC diphosphate, and the HPMPC phosphate adduct, respectively. HPMPC diphosphate inhibits herpes simplex virus-1 and -2 DNA polymerases with a lower Ki than that for DNA polymerase alpha, and enzyme inhibition is competitive in each case. The formation and the persistence of HPMPC phosphates in cells and the selective inhibition of viral DNA polymerases by HPMPC diphosphate can explain why cells pretreated with HPMPC remain refractory to viral infection even long after HPMPC is removed from the medium.
Mol Pharmacol 1992 Jan
PMID:Intracellular metabolism of the antiherpes agent (S)-1-[3-hydroxy-2-(phosphonylmethoxy)propyl]cytosine. 131 Jan 43

The carbocyclic analog of 2'-deoxyguanosine (CdG) is active against herpes simplex virus (HSV), human cytomegalovirus, and human hepatitis-B virus. In order to understand the mechanism of action of this compound against HSV, we have evaluated (a) the incorporation of [3H]CdG into viral and host DNA in HEp-2 cells infected with HSV and (b) the interaction of the 5'-triphosphate of CdG (CdG-TP) with the HSV DNA polymerase and human DNA polymerases alpha, beta, and gamma (EC 2.7.7.7). Incubation of HSV-1-infected HEp-2 cells with [3H]CdG resulted in the incorporation of CdG into both the HSV and the host cell DNA. These results indicated that CdG-TP was used as a substrate for HSV DNA polymerase and for at least one of the cellular DNA polymerases. Degradation of both viral and host DNA with micrococcal nuclease and spleen phosphodiesterase indicated that CdG was incorporated primarily into internal positions in both DNAs. The viral DNA containing CdG sedimented in neutral and alkaline sucrose gradients in the same way as did viral DNA labeled with [3H]thymidine, indicating that the HSV DNA containing CdG was similar in size to untreated HSV DNA. CdG-TP was a competitive inhibitor of the incorporation of dGTP into DNA by the HSV DNA polymerase (Ki of 0.35 microM) and the human DNA polymerase alpha (Ki of 1 microM). CdG-TP was not a potent inhibitor of either DNA polymerase beta or gamma. Using DNA-sequencing technology, CdG-TP was found to be an efficient substrate for HSV DNA polymerase. Incorporation of CdG monophosphate (CdG-MP) into the DNA by HSV DNA polymerase did not interfere with subsequent chain extension. These results suggested that the antiviral activity of CdG was due to its incorporation into the DNA and subsequent disruption of viral functions. In contrast, CdG-TP was not as good as dGTP as a substrate for DNA synthesis by DNA polymerase alpha, and incorporation of CdG-MP by DNA polymerase alpha inhibited further DNA chain elongation.
Mol Pharmacol 1992 Feb
PMID:Incorporation of the carbocyclic analog of 2'-deoxyguanosine into the DNA of herpes simplex virus and of HEp-2 cells infected with herpes simplex virus. 131 7

The relative order of 11 loci in the distal half of the short arm of the human X chromosome was examined using a panel of somatic cell hybrids containing structurally rearranged X chromosomes. The results show that the gene for phosphoribosylpyrophosphate synthetase 2 (PRPS2) is located between ZFX (zinc finger protein, X-linked) and STS (steroid sulfatase). The results also confirm the localization of ZFX distal to POLA (alpha-DNA polymerase). Previous studies have shown that STS and ZFX escape X-inactivation whereas POLA undergoes inactivation. Evaluation of PRPS2 expression in somatic cell hybrids containing inactive human X chromosomes showed that PRPS2 undergoes X-inactivation. These results provide further evidence for interspersion of loci that do and do not undergo X-inactivation on the human X chromosome.
Somat Cell Mol Genet 1992 Mar
PMID:Physical mapping of loci in the distal half of the short arm of the human X chromosome: implications for the spreading of X-chromosome inactivation. 131 58

The role of DNA polymerases in the replication of SV40 DNA was studied using a T-antigen-dependent assay supplemented with a human KB cell extract. Inhibition of DNA polymerase alpha by addition of aphidicolin or monoclonal antibodies prevented DNA synthesis, confirming the requirement for this enzyme in replication. The replication process was unaffected by ddTTP at a concentration (5 microM) inhibitory to DNA polymerases beta and gamma, however, higher concentrations of ddTTP (200 microM) caused an apparent accumulation of relaxed circular plasmid with a concomitant decrease in DNA synthesis. An analysis of this replication intermediate indicated that it was formed during the replication reaction and that the replicative cycle was nearly complete. A kinetic study of ddTTP inhibition strongly suggested DNA polymerase epsilon (PCNA-independent DNA polymerase delta) was the target of the inhibitor and that this enzyme functions during the final stages of DNA replication.
Mol Cell Biochem 1992 Mar 04
PMID:Dideoxynucleoside triphosphates inhibit a late stage of SV40 DNA replication in vitro. 131 27

The incorporation of 6-thioguanine (S6G) in place of guanine proceeds readily in DNA synthesis reactions catalyzed by mammalian and bacterial polymerases. This report summarizes the consequences of such incorporation studied to date. S6G was incorporated into one strand of a defined M13mp18 phage sequence in a (+)reaction catalyzed by the Klenow fragment of Escherichia coli DNA polymerase I. After denaturation of the newly synthesized strand (containing S6G) and annealing with a reverse (-) 32P-labeled primer, polymerization catalyzed by the Klenow enzyme as well as by human DNA polymerases alpha, gamma, and delta was slowed considerably, compared with that across the corresponding guanine-containing template. To evaluate S6G-containing DNA as a substrate for DNA ligases, two oligodeoxynucleotides (19- and 20-mers) antisense to a 40-mer were synthesized so that the 40-mer coded for guanine at the 3' terminus of the 19-mer. After annealing of the synthetic oligonucleotides to form a duplex DNA containing a one-nucleotide gap (opposite cytosine in the 40-mer), the 19-mer was extended with 2'-deoxythioguanosine 5'-triphosphate using DNA polymerase, forming a nicked duplex DNA. The abilities of T4 DNA ligase and HeLa and calf thymus DNA ligase I to join the 5'-phosphate with the 3'-S6G-OH were severely inhibited, compared with the 3'-guanine-extended control. This finding suggests that incorporation of S6G at the 3' terminus of Okazaki fragments would inhibit lagging strand DNA synthesis. In other experiments, cleavage of S6G-containing DNA by some but not all restriction endonucleases progressed poorly, compared with the control guanine-containing DNA, independently of the location of S6G at recognition or cleavage sites, as previously observed by Iwaniec et al. [Mol. Pharmacol. 39:299-306 (1991)] with a different spectrum of enzymes. These findings indicate altered DNA-protein interactions due to S6G incorporation. The poor template function of S6G-containing DNA is consistent with the known delayed cytotoxicity and DNA damage previously reported to occur in S6G-treated cells.
Mol Pharmacol 1992 Nov
PMID:Consequences of 6-thioguanine incorporation into DNA on polymerase, ligase, and endonuclease reactions. 133 62

Polymerase chain reaction (PCR) was prospectively performed with cerebrospinal fluid (CSF) from 51 patients whose CSF was available for analysis and was submitted for viral culture and/or herpes simplex virus (HSV) serology and 20 patients whose CSF was submitted exclusively to the Clinical Biochemistry Laboratory. Primers were used that flanked a 92 bp segment of the HSV DNA polymerase gene (35 cycles). Amplified products were electrophoresed on agarose gel, blotted onto nylon membrane, and probed with a 32P-labelled sequence internal to the primers. For nested PCR, 1 microliter of PCR product was amplified for an additional 35 cycles before electrophoresis and Southern blot analysis. Review of the clinical records revealed that 15 patients had central nervous system (CNS) infections. Specific HSV DNA sequences were detected in CSF specimens of three of the individuals [PCR(2), nested PCR(1)]. Two of these patients had disseminated HSV infection including encephalitis and one patient had aseptic meningitis. The diagnoses of the 12 patients with CNS infection who did not have HSV DNA detected in CSF included encephalitis [varicella-zoster virus (1), cytomegalovirus (1), Mycoplasma pneumoniae (1)], meningitis [Neisseria meningitidis (1), Coccidioides immitis (1), Enterovirus (1), aseptic meningitis (1)], varicella-zoster radiculitis (2), human immunodeficiency virus dementia (2), and transverse myelitis due to Epstein-Barr virus (1). Importantly, HSV DNA was also not detected in the CSF of the 36 patients who did not have CNS infection and 20 samples submitted exclusively to the Clinical Biochemistry Laboratory. Our findings demonstrate the utility of PCR as a rapid, non-invasive method for the routine laboratory diagnosis of CNS infection due to HSV.
Mol Cell Probes 1992 Oct
PMID:A prospective study of the polymerase chain reaction for detection of herpes simplex virus in cerebrospinal fluid submitted to the clinical virology laboratory. 133 47

Three different mutations were introduced in the polA gene of Streptococcus pneumoniae by chromosomal transformation. One mutant gene encodes a truncated protein that possesses 5' to 3' exonuclease but has lost polymerase activity. This mutation does not affect cell viability. Other mutated forms of polA that encode proteins with only polymerase activity or with no enzymatic activity could not substitute for the wild-type polA gene in the chromosome unless the 5' to 3' exonuclease domain was encoded elsewhere in the chromosome. Thus, it appears that the 5' to 3' exonuclease activity of the DNA polymerase I is essential for cell viability in S. pneumoniae. Absence of the polymerase domain of DNA polymerase I slightly diminished the ability of S. pneumoniae to repair DNA lesions after ultraviolet irradiation. However, the polymerase domain of the pneumococcal DNA polymerase I gave almost complete complementation of the polA5 mutation in Escherichia coli with respect to resistance to ultraviolet irradiation.
Mol Microbiol 1992 Oct
PMID:The 5' to 3' exonuclease activity of DNA polymerase I is essential for Streptococcus pneumoniae. 133 89

The method of analysis of low molecular weight fragments of high copied repeats of DNA hydrolysed by restriction nuclease is presented. The P labeled fragments (by means of DNA polymerase I) are electrophoresed in nondenaturing PAAG and radio-autographed. The specific band patterns are observed in region between approximately 20 and 300 bp. When studying some lizard and fish species DNA's it was shown that the patterns observed have species and genus specificity but not individual. The approach supposed can be applied to investigation of interspecies relationships, some evolutionary problems and to the studying of questions concerning the role of DNA repeats in evolution. The method is simple and comparatively cheap and may be called as "taxonomic DNA fingerprint" or "DNA taxonoprint" method.
Mol Biol (Mosk)
PMID:[Taxonometric analysis of DNA repeating elements]. 133 61

We have analyzed the CTF4 (CHL15) gene, earlier identified in two screens for yeast mutants with increased rates of mitotic loss of chromosome III and artificial circular and linear chromosomes. Analysis of the segregation properties of circular minichromosomes and chromosome fragments indicated that sister chromatid loss (1:0 segregation) is the predominant mode of chromosome destabilization in ctf4 mutants, though nondisjunction events (2:0 segregation) also occur at an increased rate. Both inter- and intrachromosomal mitotic recombination levels are elevated in ctf4 mutants, whereas spontaneous mutation to canavanine resistance was not elevated. A genomic clone of CTF4 was isolated and used to map its physical and genetic positions on chromosome XVI. Nucleotide sequence analysis of CTF4 revealed a 2.8-kb open reading frame with a 105-kDa predicted protein sequence. The CTF4 DNA sequence is identical to that of POB1, characterized as a gene encoding a protein that associates in vitro with DNA polymerase alpha. At the N-terminal region of the protein sequence, zinc finger motifs which define potential DNA-binding domains were found. The C-terminal region of the predicted protein displayed similarity to sequences of regulatory proteins known as the helix-loop-helix proteins. Data on the effects of a frameshift mutation suggest that the helix-loop-helix domain is essential for CTF4 function. Analysis of sequences upstream of the CTF4 open reading frame revealed the presence of a hexamer element, ACGCGT, a sequence associated with many DNA metabolism genes in budding yeasts. Disruption of the coding sequence of CTF4 did not result in inviability, indicating that the CTF4 gene is nonessential for mitotic cell division. However, ctf4 mutants exhibit an accumulation of large budded cells with the nucleus in the neck. ctf4 rad52 double mutants grew very slowly and produced extremely high levels (50%) of inviable cell division products compared with either single mutant alone, which is consistent with a role for CTF4 in DNA metabolism.
Mol Cell Biol 1992 Dec
PMID:CTF4 (CHL15) mutants exhibit defective DNA metabolism in the yeast Saccharomyces cerevisiae. 134 Nov 95

The polymerase chain reaction (PCR) uses in vitro enzymatic synthesis to amplify specific DNA sequences. PCR amplification can produce approximately 100 billion copies of one molecule of DNA in a few hours. PCR has revolutionized research in the biological sciences and medicine, and has influenced criminology and law. Several major scientific discoveries, including purification of DNA polymerase and elucidation of the mechanism of DNA replication, were essential for development of the present PCR technology. An overview of these discoveries and early work on in vitro DNA synthesis are presented. Basic PCR methodology, instrumentation, advanced PCR techniques, and applications are also discussed in this review. Several new amplification systems are mentioned. PCR is an extremely important and simple technology for research and diagnostic analyses of DNA and RNA. PCR technology and other amplification procedures will continue to produce novel applications in basic research and clinical medicine.
Diagn Mol Pathol 1992 Mar
PMID:The polymerase chain reaction. History, methods, and applications. 134 55


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