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 distribution of DNA adducts formed by the two main components, aristolochic acid I (AAI) and aristolochic acid II (AAII), of the carcinogenic plant extract aristolochic acid (AA) was examined in a plasmid containing exon 2 of the mouse c-H-ras gene by a polymerase arrest assay. AAI and AAII were reacted with plasmid DNA by reductive activation and the resulting DNA adducts were identified as the previously characterized adenine adducts (dA-AAI and dA-AAII) and guanine adducts (dG-AAI and dG-AAII) by the (32)P-post-labeling method. In addition, a structurally unknown adduct was detected in AAII-modified DNA and shown to be derived from reaction with cytosine (dC-AAII). Sites at which DNA polymerase progress along the template was blocked were assumed to be at the nucleotide 3' to the adduct. Polymerase arrest spectra showed a preference for reaction with purine bases in the mouse H-ras gene for both activated compounds, consistent with previous results that purine adducts are the principal reaction products of AAI and AAII with DNA. Despite the structural similarities among AAI-DNA and AAII-DNA adducts, however, the polymerase arrest spectra produced by the AAs were different. According to the (32)P-post-labeling analyses reductively activated AAI showed a strong preference for reacting with guanine residues in plasmid DNA, however, the polymerase arrest assay revealed arrest sites preferentially at adenine residues. In contrast, activated AAII reacted preferentially with adenine rather than guanine residues and to a lesser extent with cytosine but DNA polymerase was arrested at guanine as well as adenine and cytosine residues with nearly the same average relative intensity. Thus, the polymerase arrest spectra obtained with the AA-adducted ras sequence do not reflect the DNA adduct distribution in plasmid DNA as determined by (32)P-post-labeling. Arrest sites of DNA polymerase associated with cytosine residues confirmed the presence of a cytosine adduct in DNA modified by AAII. For both compounds adduct distribution was not random; instead, regions with adduct hot spots and cold spots were observed. Results from nearest neighbor binding analysis indicated that flanking pyrimidines displayed the greatest effect on polymerase arrest and therefore on DNA binding by AA.
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PMID:Using polymerase arrest to detect DNA binding specificity of aristolochic acid in the mouse H-ras gene. 1065 63

As a result of our increased understanding of the human genome, and the functional interrelationships of gene products with each other and with the environment, it is becoming increasingly evident that many human diseases are influenced by heritable alterations in the structure or function of genes. Significant advances in research methods and newly emerging partnerships between private and public sector interests are creating new possibilities for utilization of genetic information for the diagnosis and treatment of human diseases. The availability and application of genetic information to the understanding of normal and abnormal human growth and development are fundamentally changing the way we approach the study of human diseases. As a result, the issues and principles of medical genetics are coming to bear across all disciplines of health care. In this review, we discuss some of the potential applications of human molecular genetics for the diagnosis and treatment of oral diseases. This discussion is presented in the context of the ongoing technological advances and conceptual changes that are occurring in the field of medical genetics. To realize the promise of this new molecular genetics, we must be prepared to foresee the possibilities and to incorporate these newly emergent technologies into the evolving discipline of dentistry. By using examples of human conditions, we illustrate the broad application of this emerging technology to the study of simple as well as complex genetic diseases. Throughout this paper, we will use the following terminology: Penetrance--In a population, defined as the proportion of individuals possessing a disease-causing genotype who express the disease phenotype. When this proportion is less than 100%, the disease is said to have reduced or incomplete penetrance. Polymerase chain reaction (PCR)--A technique for amplifying a large number of copies of a specific DNA sequence flanked by two oligonucleotide primers. The DNA is alternately heated and cooled in the presence of DNA polymerase and free nucleotides, so that the specified DNA segment is denatured, hybridized with primers, and extended by DNA polymerase. MIM--Mendelian Inheritance in Man catalogue number from V. McKusick's Mendelian Inheritance in man (OMIM, 1998).
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PMID:The impact of molecular genetics on oral health paradigms. 1068

The BRCA1 COOH terminus (BRCT) motif is present in many nuclear proteins that contribute to cell cycle regulation or DNA repair. Polymerase chain reaction-based screening with degenerate primers targeted to the BRCT motif resulted in the isolation of a human cDNA for a previously unidentified DNA polymerase (designated DNA polymerase beta2) that is closely related to DNA polymerase beta (Pol beta). The predicted Pol beta2 protein contains a BRCT motif in its NH(2)-terminal region; its COOH-terminal region exhibits 33% sequence identity to a corresponding region of human Pol beta. The Pol beta2 gene is expressed in a tissue-specific manner, with transcripts being most abundant in testis. A fusion construct comprising Pol beta2 and green fluorescent protein exhibited a predominantly nuclear localization in transfected HeLa cells. Recombinant human Pol beta2 from insect cells exhibited substantial DNA polymerase activity, but it did not possess terminal deoxyribonucleotidyl transferase activity. A truncated Pol beta2 mutant lacking the BRCT motif retained substantial DNA polymerase activity, whereas a mutant Pol beta2 with two alanine point mutations within the DNA polymerase active site did not. These results indicate that Pol beta2 is a Pol beta-related DNA polymerase with a BRCT motif that is dispensable for its polymerase activity.
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PMID:Identification and characterization of human DNA polymerase beta 2, a DNA polymerase beta -related enzyme. 1088 91

Fever, cutaneous rash, and hepatitis-for which an infectious cause was suspected-developed in an Italian patient with non-Hodgkin lymphoma after autologous peripheral blood stem cell (PBSC) transplantation. Polymerase chain reaction (PCR) with degenerate primers for the highly conserved DNA polymerase gene of herpesviruses detected herpesvirus sequences 100% identical to human herpesvirus-8 (HHV-8) in serial cell-free serum samples, collected immediately before or concomitant with the occurrence of clinical symptoms; no other common infections were documented. The presence of the HHV-8 genome (clade C) was confirmed by PCR with HHV-8-specific primers for orf 26 and orf-K1. HHV-8 viremia was undetectable either before transplantation or when the patient was clinically asymptomatic. Semiquantitative PCR analysis showed variations of the viral load correlating with the clinical status. Anti-HHV-8 antibodies were detected before and after transplantation by an immunofluorescence assay for lytic antigens. Active HHV-8 infection may be associated with nonmalignant illness after PBSC/bone marrow transplantation.
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PMID:Nonmalignant disease associated with human herpesvirus 8 reactivation in patients who have undergone autologous peripheral blood stem cell transplantation. 1100 82

Polymerase zeta (Pol zeta) is an error-prone DNA polymerase [1], which in yeast is involved in trans-lesion synthesis (TLS) and is responsible for most of the ultraviolet (UV) radiation-induced and spontaneous mutagenesis [2-4]. Pol zeta consists of three subunits: REV1, a deoxycytidyl-transferase [5]; REV7, of unclear function [6]; and REV3, the catalytic subunit. REV3 alone is sufficient to carry out TLS, but association with REV1 and REV7 enhances its activity [5, 7]. Experiments using human cells treated with UV radiation indicate also that mammalian Pol zeta is involved in TLS [7]. The peculiar mutagenic activity of Pol zeta [4,7,8] suggests a possible role in somatic hypermutation of immunoglobulin (Ig) genes [9]. Here, we report that, unlike in yeast where the REV3 gene is not essential for life [4], disruption of the mouse homologue (Rev3l) resulted in early embryonic lethality. In Rev3l(-/-) embryos, no haematopoietic cells other than erythrocytes could be identified in the yolk sac. Rev3l(-/-) haematopoietic precursors were unable to expand in vitro and no haematopoietic cells could be derived from the intraembryonic haematogenic compartment (splanchnopleura). Fibroblasts could not be derived from the Rev3l(-/-) embryos, and Rev3l(-/-) embryonic stem (ES) cells could not be obtained. This is the first evidence that an enzyme involved in TLS is critical for mammalian development.
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PMID:Disruption of the Rev3l-encoded catalytic subunit of polymerase zeta in mice results in early embryonic lethality. 1105 Mar 93

The carcinogenic plant extract aristolochic acid (AA) is thought to be the major causative agent in the development of urothelial carcinomas found in patients with Chinese herb nephropathy (CHN). These carcinomas are associated with overexpression of p53, suggesting that the p53 gene is mutated in CHN-associated urothelial malignancy. To investigate the relation between AA-DNA adduct formation and possible p53 mutations, we mapped the distribution of DNA adducts formed by the two main components of AA, aristolochic acid I (AAI) and aristolochic acid II (AAII) at single nucleotide resolution in exons 5-8 of the human p53 gene in genomic DNA. To this end, an adduct-specific polymerase arrest assay combined with a terminal transferase-dependent PCR (TD-PCR) was used to amplify DNA fragments. AAI and AAII were reacted with human mammary carcinoma (MCF-7) DNA in vitro and the major DNA adducts formed were identified by the (32)P-postlabeling method. These adducted DNAs were used as templates for TD-PCR. Sites at which DNA polymerase progress along the template was blocked were assumed to be at the nucleotide 3' to the adduct. Polymerase arrest spectra thus obtained showed a preference for reaction with purine bases in the human p53 gene for both activated compounds. For both AAs, adduct distribution was not random; the strongest signals were seen at codons 156, 158-159 and 166-167 for exon 5, at codons 196, 198-199, 202, 209, 214-215 and 220 for exon 6, at codons 234-235, 236-237 and 248-249 for exon 7 and at codons 283-284 and 290-291 for exon 8. Overall guanines at CpG sites in the p53 gene that correspond to mutational hotspots observed in many human cancers seem not to be preferential targets for AAI or II. We compared the AA-DNA binding spectrum in the p53 gene with the p53 mutational spectrum of urothelial carcinomas found in the human mutation database. No particular pattern of polymerase arrest was found that predicts AA-specific mutational hotspots in urothelial tumors of the current p53 database. Thus, AA is not a likely cause of non-CHN-related urothelial tumors.
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PMID:Sequence-specific detection of aristolochic acid-DNA adducts in the human p53 gene by terminal transferase-dependent PCR. 1115 51

Two immature female bottlenose dolphins (Tursiops truncatus) were found stranded on the Atlantic coast of the USA. Necropsy and histopathologic examination of both dolphins demonstrated acute necrotizing lesions in multiple organ systems. Commonly seen in these lesions were cells with enlarged nuclei that contained single 4 to 6 microm diameter homogeneous eosinophilic inclusion bodies that were often surrounded by a clear halo. Ultrastructural examination revealed that intranuclear inclusions contained 90 to 110 nm diameter viral particles with electron-dense cores and hexagonal profiles. Viral particles were also present in the cytoplasm, and these were surrounded by variably electron-dense envelopes. Enveloped virions were 140 nm in diameter. Polymerase chain reactions targeting the DNA polymerase and terminase genes of herpesviruses were carried out on unfixed tissues of both animals, and analysis of the DNA products indicated the presence of two novel alphaherpesviruses. The gross, histologic, ultrastructural, and molecular genetic findings indicate disseminated herpesviral infections, and support the conclusion that the alphaherpesviruses caused the deaths of the two dolphins. This is the first report of disseminated herpesviral infection in cetaceans.
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PMID:Two novel alphaherpesviruses associated with fatal disseminated infections in Atlantic bottlenose dolphins. 1131 Aug 80

Replication of kinetoplast DNA minicircles in Crithidia fasciculata occurs by a unidirectional mechanism involving continuous synthesis of one strand (L strand) and discontinuous synthesis of the complementary strand (H strand). L-strands are initiated by RNA priming at alternate origins (A and B) resulting in daughter molecules with a single nick or gap in the L strand at either ori A or ori B. Some of the gapped molecules contain ribonucleotides at the 5' side of the gap. We have investigated the ability of recombinant forms of kinetoplast replication proteins, DNA polymerase beta and structure specific endonuclease 1, to repair gaps in a model minicircle substrate. Structure specific endonuclease 1 was shown to efficiently remove all ribonucleotides from the 5' side of the model substrate by stepwise cleavage of the RNA primer. Polymerase beta was then able to extend the 3' terminus of the gap to yield a nicked molecule capable of covalent joining by a DNA ligase. These results demonstrate that the nuclease and polymerase enzymes present at antipodal protein complexes flanking the kinetoplast disk are capable of complete RNA primer removal and subsequent gap filling of newly synthesized minicircle L strands.
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PMID:RNA primer removal and gap filling on a model minicircle replication intermediate. 1137 40

The DNA Polymerase III holoenzyme forms initiation complexes on primed DNA in an ATP-dependent reaction. We demonstrate that the nonhydrolyzable ATP analog, ATP gamma S, supports the formation of an isolable leading strand complex that loads and replicates the lagging strand only in the presence of ATP, beta, and the single-stranded DNA binding protein. The single endogenous DnaX complex within DNA polymerase III holoenzyme assembles beta onto both the leading and lagging strand polymerases by an ordered mechanism. The dimeric replication complex disassembles in the opposite order from which it assembled. Upon ATP gamma S-induced dissociation, the leading strand polymerase is refractory to disassembly allowing cycling to occur exclusively on the lagging strand. These results establish holoenzyme as an intrinsic asymmetric dimer with distinguishable leading and lagging strand polymerases.
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PMID:The DNA polymerase III holoenzyme: an asymmetric dimeric replicative complex with leading and lagging strand polymerases. 1143 88

The crystal structure of the DNA polymerase encoded by gene 5 of bacteriophage T7, in a complex with its processivity factor, Escherichia coli thioredoxin, a primer-template, and an incoming deoxynucleoside triphosphate reveals a putative hydrogen bond between the C-terminal residue, histidine 704 of gene 5 protein, and an oxygen atom on the penultimate phosphate diester of the primer strand. Elimination of this electrostatic interaction by replacing His(704) with alanine renders the phage nonviable, and no DNA synthesis is observed in vivo. Polymerase activity of the genetically altered enzyme on primed M13 DNA is only 12% of the wild-type enzyme, and its processivity is drastically reduced. Kinetic parameters for binding a primer-template (K(D)(app)), nucleotide binding (K(m)), and k(off) for dissociation of the altered polymerase from a primer-template are not significantly different from that of wild-type T7 DNA polymerase. However, the decrease in polymerase activity is concomitant with increased hydrolytic activity, judging from the turnover of nucleoside triphosphate into the corresponding nucleoside monophosphate (percentage of turnover, 65%) during DNA synthesis. Biochemical data along with structural observations imply that the terminal amino acid residue of T7 DNA polymerase plays a critical role in partitioning DNA between the polymerase and exonuclease sites.
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PMID:Role of the C-terminal residue of the DNA polymerase of bacteriophage T7. 1145 60


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