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)

Previously we raised a rabbit monospecific antibody (C2003) against a synthetic peptide derived from a sequence within the C-terminal portion of the reverse transcriptase (RT) of the human immunodeficiency virus type 1 (HIV-1). This sequence is found to be conserved in the predicted amino acid sequence of a related lentivirus, the equine infectious anemia virus (EIAV). It was previously determined that the C2003 antibody could cross-react with native EIAV RT and directly inhibit the DNA polymerase activity of the enzyme. We have now fractionated EIAV RT by immunoaffinity chromatography with immobilized C2003 antibody. The procedure yielded an equimolar mixture of two proteins of 66 and 51 kDa associated with both DNA polymerase and RNase H activities. When the EIAV RT proteins were examined by in situ activity gel assays, polymerase activity was found to be principally associated with the 66-kDa component. The fidelity of DNA synthesis by EIAV RT was found to be equivalent to that of HIV-1 RT and lower than that of AMV RT. These observations indicate that the RTs of EIAV and HIV-1 share similar structural and functional properties.
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PMID:Purification and partial characterization of equine infectious anemia virus reverse transcriptase. 171 86

The RNA- and DNA-dependent DNA polymerase activities of two point mutants of HIV-1 reverse transcriptase lacking ribonuclease H activity have been compared to the wild-type enzyme activities using substrates consisting of an oligodeoxynucleotide primer hybridized to either a RNA or a DNA template. The RNase H phenotype had a negligible effect on the steady-state kinetics and processivity of reverse transcription of a homopolymer template-primer [poly(A).oligo(dT)]. However, analysis of the distribution of DNA products indicated that the ability of the mutants to reverse-transcribe a specifically primed 345-nucleotide heteropolymeric RNA template derived from the gag region of HIV-1 was impaired relative to the wild-type enzyme. Although the wild-type and mutant enzymes shared the same pause sites of synthesis along the RNA template, certain prematurely terminated nascent primer chains were poorly extended by the mutant enzymes and hence accumulated, suggesting that a catalytically functional RNase domain facilitated reinitiation of DNA synthesis at specific pause sites along a heteropolymer template. In contrast, the processivity and product distribution of DNA synthesis directed by a heteropolymer gag DNA template of the same nucleotide sequence were not significantly influenced by the RNase H phenotype of the mutants.
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PMID:Analysis of the RNA- and DNA-dependent DNA polymerase activities of point mutants of HIV-1 reverse transcriptase lacking ribonuclease H activity. 171 22

The rational design of antiviral agents targeting the reverse transcriptase (RT) of the human immunodeficiency virus (HIV) would greatly benefit from a more intimate knowledge of the structure of RT. Until now, the degree of sequence similarity between RT and E. coli DNA polymerase I (Pol I) has been thought to be confined to several small regions, suggesting little basis for homology molecular modeling. However, we have found that a region in the C terminal of the RT polymerase domain is homologous to a central region of Pol I that lies between the universal polymerase motifs A and C (specifically, helices N-O-P of the Pol I crystal structure); a single transposition closely aligns the RT and Pol I genes, revealing a similar domain structure with 20% residue identity, as well as the possible structural correlates of several RNA-dependent polymerase motifs. The RT from Myxococcus xanthus (a bacterium believed to have diverged from other species 2 billion years ago), if similarly transposed, shows homology to both HIV-1 and E. coli, suggesting the possibility of a very ancient divergence between the RT and Pol I polymerase genes. A second even more significant match to this E. coli region was found in the retroviral ribonuclease H (RNase H) domain, and corresponds precisely to a region that has been aligned by previous investigators with the E. coli RNase H, suggesting that Pol I helices O and P are homologous to helices A and D of the RNase H crystal structure, respectively. These results are consistent with a modular theory of molecular evolution.
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PMID:A transposition of the reverse transcriptase gene reveals unexpected structural homology to E. coli DNA polymerase I. 172 84

We have constructed a series of plasmids which, when introduced into Escherichia coli, induce the overexpression of soluble wild-type and mutated forms of the reverse transcriptases (RTs) from human immunodeficiency viruses types 1 and 2 (HIV-1 and HIV-2, respectively). These proteins were analyzed previously for their RNA-dependent DNA polymerase (RDDP) and ribonuclease H (RNase H) activities. In the present study we assayed the different mutant RTs for their DNA-dependent DNA polymerase (DDDP) activity, employing an in situ polyacrylamide gel activity assay. The results indicate that both the RDDP and DDDP catalytic functions of HIV-1 RT mutants are affected similarly by mutations suggesting a high degree of overlap between the catalytic domains involved in both activities. Contrariwise, many of the HIV-2 RT mutants display no correlation between these two DNA polymerase activities, that is, the DDDP activity was not affected by the mutations introduced in the native enzyme in contrast to the RDDP activity. We were thus able to generate mutants of HIV-2 RT that unlike the wild-type RT, are capable of transcribing only DNA and not RNA. The disparity in mutational-catalytic relations between the two HIV-related RTs may reflect a possible difference in the structure and folding properties of the two proteins.
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PMID:The DNA-dependent and RNA-dependent DNA polymerase activities of the reverse transcriptases of human immunodeficiency viruses types 1 and 2. 172 5

Improvement of a cDNA synthesis procedure using a single stranded (ss) vector primer [Bellemare et al., Gene 52 (1987) 11-19] is reported. This vector (pPBS27), upon linearization with XbaI using an appropriate restriction site-directed fragment, releases a thymidilic tail used to prime cDNA synthesis. DNA polymerase I and RNase H replace the RNA strand and replicate the vector before double-stranded (ds) blunt-end ligation with T4 DNA ligase. More than 10(7) cfu/microgram of vector can be obtained with an efficient transformation protocol using either globin-encoding or 7.5-kb poly(A)-tailed RNA. This improved cloning method is easier, faster and a few hundred times more efficient than the original procedure as it involves ds rather than ss DNA for transformation.
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PMID:High-yield method for directional cDNA library construction. 184 63

pBR322 DNA can be replicated via a DNA A-dependent pathway mediated by its binding to the two DNA A-binding sites (dnaA boxes) present near the plasmid origin. DNA synthesis requires the transcription of RNA II (the leading-strand primer precursor) to generate a specific unwound structure in the region containing the dnaA boxes. In this structure, the DNA containing the dnaA boxes can take the form of either a RNA II-parental H strand pBR322 DNA hybrid opposed by the displaced parental L strand (in the absence of RNase H and DNA polymerase I), or a nascent leading strand-parental H strand DNA duplex opposed by the displaced parental L strand (in the presence of RNase H and DNA polymerase I). These findings defined three types of potential sites for productive DNA A binding: (i) the displaced parental L single strand, (ii) a hairpin formed by the inverted repeat of the two dnaA boxes, and (iii) either the RNA-DNA duplex or the nascent leading strand-parental DNA duplex. By using a combination of: (i) inhibition of the replication of a plasmid carrying oriC by oligonucleotides of various dnaA box sequences and conformation, (ii) a gel mobility shift assay to measure DNA A binding to the same oligonucleotide substrates, (iii) replication of pBR322 DNA templates with either one or no dnaA box, and (iv) photocross-linking to demonstrate DNA A binding to an RNA-DNA hybrid, evidence is presented here that DNA A-mediated pBR322 DNA replication proceeds by a mechanism in which DNA A binds to the duplex side of the unwound origin structures and loads the DNA B protein in trans to the displaced parental L strand DNA.
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PMID:Mechanism of DNA A protein-dependent pBR322 DNA replication. DNA A protein-mediated trans-strand loading of the DNA B protein at the origin of pBR322 DNA. 191 6

To correlate the hepatitis B virus P gene with the enzymatic activities predicted to participate in hepadnavirus reverse transcription, a series of P gene mutants containing missense mutations, in-phase insertions, and in-phase deletions was constructed by site-directed mutagenesis. These mutants were tested in the context of otherwise intact hepatitis B virus genomes for the ability to produce core particles containing the virus-associated polymerase activity. The results obtained suggest that the P protein consists of three functional domains and a nonessential spacer arranged in the following order: terminal protein, spacer, reverse transcriptase/DNA polymerase, and RNase H. The first two domains are separated by a spacer region which could be deleted to a large extent without significant loss of endogenous polymerase activity. In cotransfection experiments, all P gene mutants could be complemented in trans by constructs expressing the wild-type gene product but not by a second P gene mutant. This indicates that the multifunctional P gene is expressed as a single translational unit and independent of the core gene and furthermore that the gene product is freely diffusible and not processed before core assembly.
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PMID:Mutational analysis of the hepatitis B virus P gene product: domain structure and RNase H activity. 215 28

Two forms of DNA primase stimulatory factor have been purified from mouse FM3A cells and shown to have RNase H activity. One of the factors, which consists of three polypeptides of 42,000, 41,000, and 27,000 daltons, was characterized in its properties as RNase H and DNA primase stimulatory factor. The nucleolytic activity of the factor specifically digested the RNA component of RNA-DNA hybrids in an endonucleolytic manner. The stimulation by the factor was observed in DNA synthesis by DNA primase-DNA polymerase alpha complex on unprimed DNA templates, and the DNA chains synthesized under these conditions in the presence of the factor were much shorter than those synthesized in its absence. The stimulatory effect of the factor on DNA primase activity was directly confirmed with DNA primase dissociated from DNA polymerase alpha by the observation of the increase in the number of synthesized oligoribonucleotides. The primer RNA synthesis by DNA primase-DNA polymerase alpha complex under the condition where DNA synthesis occurred was also significantly stimulated by the factor. Furthermore, under these conditions RNA primers were removed from DNA chains by the RNase H activity of the factor.
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PMID:DNA primase stimulatory factor from mouse FM3A cells has an RNase H activity. Purification of the factor and analysis of the stimulation. 216 40

The analysis of the deduced amino acid sequence of the herpes simplex virus type 1 (HSV-1) DNA polymerase reported here suggests that the polymerase structure consists of domains carrying separate biological functions. The HSV-1 enzyme is known to possess 5'-3'-exonuclease (RNase H), 3'-5'-exonuclease, and DNA polymerase catalytic activities. Sequence analysis suggests an arrangement of these activities into distinct domains resembling the organization of Escherichia coli polymerase I. In order to more precisely define the structure and C-terminal limits of a putative catalytic domain responsible for the DNA polymerization activity of the HSV-1 enzyme, we have undertaken in vitro mutagenesis and computer modeling studies of the HSV-1 DNA polymerase gene. Sequence analysis predicts that the major DNA polymerization domain of the HSV-1 enzyme will be contained between residues 690 and 1100, and we present a three-dimensional model of this region, on the basis of the X-ray crystallographic structure of the E. coli polymerase I. Consistent with these structural and modeling studies, deletion analysis by in vitro mutagenesis of the HSV-1 DNA polymerase gene expressed in Saccharomyces cerevisiae has confirmed that certain amino acids from the C terminus (residues 1073 to 1144 and 1177 to 1235) can be deleted without destroying HSV-1 DNA polymerase catalytic activity and that the extreme N-terminal 227 residues are also not required for this activity.
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PMID:Structure-function studies of the herpes simplex virus type 1 DNA polymerase. 216 83

A sedimentable complex of enzymes for DNA synthesis was partially purified from the combined low-salt nuclear extract-postmicrosomal supernatant solution of HeLa cell homogenates by poly(ethylene glycol) precipitation in the presence of 2 M KCl, discontinuous gradient centrifugation, Q-Sepharose chromatography, and velocity gradient centrifugation. In addition to the previously described 640-kDa multiprotein DNA polymerase alpha-primase complex [Vishwanatha et al. (1986) J. Biol. Chem. 261, 6619-6628], the enzyme complex also has associated topoisomerase I, DNA-dependent ATPase, RNase H, DNA ligase, a simian virus 40 origin recognition, dA/dT sequence binding protein [Malkas & Baril (1989) Proc. Natl. Acad. Sci. U.S.A. 86, 70-74], and proliferating cell nuclear antigen. Essentially all of the T antigen dependent simian virus 40 in vitro replication activity in the combined nuclear extract-postmicrosomal supernatant solution resides with the sedimentable complex of enzymes for DNA synthesis. Sedimentation analysis on a 10-35% glycerol gradient in the presence of 0.5 M KCl indicates that the enzyme complex is 21S. The associated enzymes for DNA synthesis and in vitro simian virus 40 replication activity cofractionate throughout the purification of the 21S complex. The DNA polymerase and in vitro simian virus 40 replication activities are both inhibited by monoclonal antibody (SJK 132-20) to human DNA polymerase alpha and by 5-10 microM butylphenyl-dGTP, indicating that the association of DNA polymerase alpha with the 21S enzyme complex is essential for the initiation of SV40 DNA replication in vitro.
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PMID:A 21S enzyme complex from HeLa cells that functions in simian virus 40 DNA replication in vitro. 216 68


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