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 major herpes simplex virus DNA-binding protein, ICP8, was purified from cells infected with the herpes simplex virus type 1 temperature-sensitive strain tsHA1. tsHA1 ICP8 bound single-stranded DNA in filter binding assays carried out at room temperature and exhibited nonrandom binding to single-stranded bacteriophage fd DNA circles as determined by electron microscopy. The filter binding assay results and the apparent nucleotide spacing of the DNA complexed with protein were identical, within experimental error, to those observed with wild-type ICP8. Thermal inactivation assays, however, showed that the DNA-binding activity of tsHA1 ICP8 was 50% inactivated at approximately 39 degrees C as compared with 45 degrees C for the wild-type protein. Both wild-type and tsHA1 ICP8 were capable of stimulating viral DNA polymerase activity at permissive temperatures. The stimulatory effect of both proteins was lost at 39 degrees C.
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PMID:In vitro characterization of a thermolabile herpes simplex virus DNA-binding protein. 301 19

Two herpes simplex virus (HSV) intertypic recombinants were isolated with genomes composed entirely of HSV-2(186) nucleotide sequences except for a 6.0 kb segment of HSV-1(17) DNA positioned between 0.40 and 0.44 map units. Following corneal infection of mice, HSV-1(17) and the two intertypic recombinants spread from infected eyes into the central nervous system and induced a fatal encephalitis. Ocular infection with the HSV-2(186) parent did not lead to detectable amounts of virus in the brain, and none of the mice developed encephalitis. The 6.0 kb HSV-1(17) DNA inserted within the genome of the two intertypic recombinants contained nucleotide sequences involved in DNA replication. These include the HSV-1(17) oriL, the HSV-1(17) gene for DNA polymerase and portions of the HSV-1(17) gene coding for DNA-binding protein ICP8. Thus, our results indicate that the difference in the capacity of HSV-1(17) and HSV-2(186) to spread from the cornea into the CNS is determined solely by nucleotide sequences associated with DNA replication.
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PMID:Nucleotide sequences important in DNA replication are responsible for differences in the capacity of two herpes simplex virus strains to spread from cornea to central nervous system. 303 Jun 43

The effect of short-term herpes simplex virus type 1 (HSV-1) infection on chromosomes of human diploid fibroblasts was examined. In addition to chromosomal breaks, gaps and pulverization, three kinds of cytogenetic damage (double minutes, polyploidy and endoreduplication) not yet reported following productive infection with HSV or other animal viruses were frequently observed. Consistent with previous studies suggesting that the expression of immediate-early and/or early viral gene products is required for the induction of chromosomal damage, was the observation that cells infected at the nonpermissive temperature with HSV-1 temperature-sensitive mutants defective in the gene for the immediate-early transcriptional regulatory protein, ICP4, and three early viral gene products--DNA polymerase (pol), the major HSV DNA-binding protein (ICP8) and an HSV-2 mutant defective in alkaline nuclease--exhibited altered patterns of chromosomal damage relative to the effects of wild-type virus on infected cells. These findings suggest a direct or indirect role for all four gene products in the induction of chromosomal damage. In cells infected with wild-type virus for 4 h or longer, HSV proved to be a more potent mitotic arresting agent than colcemid. Moreover, studies with selected mutants indicate that HSV pol specifically may be involved in mitotic arrest. Additionally, in cells infected at the non-permissive temperature with a pol mutant, the number of polyploid metaphases was reduced 4-fold relative to that seen in wild-type virus-infected cells suggesting a role for HSV pol in the amplification of cellular DNA.
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PMID:Herpes simplex virus gene products involved in the induction of chromosomal aberrations. 303 32

The mechanism of enhancement of DNA polymerase activity by the murine DNA-binding protein factor D was investigated. Extension by Escherichia coli DNA polymerase I and calf thymus DNA polymerase-alpha of 5'-32P-labeled oligodeoxynucleotide primers that are complementary to poly(dT) or to bacteriophage M13 DNA was measured in the absence or presence of factor D. With 5'-[32P](dA)9.poly(dT), factor D enables E. coli polymerase I to fill approximately 15-nucleotide gaps between adjacent primers; whereas in the absence of the stimulatory protein, poly(dT) is not copied significantly. In order to study the nucleotide specificity of synthesis enhancement, we used M13mp10 DNA containing 4 consecutive thymidine residues downstream from the 3-hydroxyl terminus of an oligonucleotide primer. Upon addition of factor D, both polymerase I and polymerase-alpha can traverse this sequence more efficiently and thus generate longer DNA products. Densitometric analysis of nonextended and elongated 5'-32P-labeled M13 primer indicates that, without changing the frequency of primer utilization, factor D enhances the activity of these DNA polymerases by increasing their apparent processivity. By positioning oligonucleotide primers 4, 8, and 12 bases upstream from the (dT)4 template sequence, we show that the enhancement of synthesis by factor D is independent of the position of the oligothymidine cluster. We hypothesize that factor D interacts with oligo(dT).oligo(dA) domains in DNA to alter their conformation, which may normally obstruct the progression of DNA polymerases.
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PMID:The DNA sequence specificity of stimulation of DNA polymerases by factor D. 329 45

Replication of bacteriophage T7 DNA initiates in vivo at an origin located 15% of the distance from the genetic left end of the chromosome. Bidirectional DNA synthesis from this site results in complete replication of the chromosome. The combination of T7 RNA polymerase, T7 DNA polymerase, and T7 gene 4 protein initiates DNA synthesis in vitro within the cloned origin sequence (Fuller, C. W., and Richardson, C. C. (1985) J. Biol. Chem. 260: 3185-3196). DNA synthesis is primed by T7 RNA polymerase transcripts, and proceeds in the same direction (rightward) as transcription to yield partially replicated Y-form DNA molecules. The DNA product of in vitro synthesis (Y-form DNA) has been characterized by electron microscopic, sedimentation, and gel electrophoretic analyses. These studies show that Y-form DNA is the product of unidirectional replication of both leading and lagging strands from the origin to the right-hand end of the template. The inclusion of either Escherichia coli single-stranded DNA-binding protein or the functionally similar T7 gene 2.5 protein results in marked stimulation of bidirectional synthesis. Studies using purified Y-form DNA provide direct evidence that this species is an intermediate in the complete replication of the linear template. Purified Y-form DNA is converted to linear DNA in a reaction catalyzed by T7 DNA polymerase, T7 gene 4 protein, and single-stranded DNA-binding protein. Y-form DNA is a competent, transient intermediate during the bidirectional replication of linear DNA molecules and DNA-binding protein is essential to initiate leftward synthesis.
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PMID:Initiation of DNA replication at the primary origin of bacteriophage T7 by purified proteins. Initiation of bidirectional synthesis. 403 7

A DNA-binding protein has been purified from Escherichia coli infected with bacteriophage T7 by DNA-cellulose chromatography. The protein is absent in uninfected cells. The purified protein has a molecular weight of 31,000 and binds strongly and preferentially to single-stranded DNA. In vitro studies show that this protein can stimulate the rate of polymerization catalyzed by the T7-induced DNA polymerase 10-15 times under conditions where the polymerase is unable to effectively use a single-stranded template. The degree of stimulation is dependent upon the ratio of binding protein to DNA template and is independent of polymerase concentration. The observed stimulation is specific for the T7 DNA polymerase in that addition of the protein to reactions catalyzed by E. coli DNA polymerases I, II, or III or T4 DNA polymerase is without effect.
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PMID:A DNA-binding protein induced by bacteriophage T7. 457 45

The interaction of the herpes simplex virus type 1 (HSV-1) major DNA-binding protein, infected-cell polypeptide 8 (ICP8), with nucleic acids has been examined by a filter-binding assay and electron microscopy. Filter-binding assays done over a broad pH range indicated that the optimum pH for the protein-DNA interaction is approximately 7.6. Heat inactivation studies showed that ICP8 is stable at temperatures up to 40 degrees C with a rapid loss of binding activity on incubation at 45 degrees C and above. Competition binding experiments have established the following relative affinities of ICP8 for the following nucleic acids: single-stranded HSV-1 DNA congruent to bacteriophage fd DNA greater than polyriboadenylate much greater than double-stranded HSV-1 DNA congruent to d(pCpT)5. Observation of negatively stained ICP8-single-stranded DNA complexes indicated that ICP8 binds along the length of the DNA in a regular repeating fashion. The average width of these complexes is 9.3 +/- 0.8 nms. Finally. Finally, addition of purified ICP8 to HSV-1 DNA polymerase reactions resulted in a stimulation of the viral polymerase activity.
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PMID:Interaction with nucleic acids and stimulation of the viral DNA polymerase by the herpes simplex virus type 1 major DNA-binding protein. 609 4

Monoclonal antibodies directed against several herpes simplex virus (HSV)-induced DNA-binding proteins were used to investigate protein interactions in HSV-infected cells. Q1 monoclonal antibody, which is specific for the HSV-induced alkaline nuclease, when used in an immunoadsorbant column resulted in the purification of the alkaline nuclease, to which large quantities of the major DNA-binding protein were bound. Conversely, when a monoclonal antibody to the major DNA-binding protein was used in affinity chromatography other polypeptides (including the DNA polymerase and alkaline nuclease) were eluted in addition to the major DNA-binding protein. Similar results were obtained when the experiment was performed using a monoclonal antibody to another HSV-2 DNA-binding protein. These results suggest the possibility that these polypeptides interact as part of the HSV DNA replication complex, and this hypothesis is discussed.
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PMID:Interactions between herpes simplex virus DNA-binding proteins. 609 15

Protein factors have been isolated from HeLa cells and from calf thymus which are able to specifically stimulate DNA polymerase alpha in vitro on templates which mimic the replication fork. One factor, extracted from HeLa cells, is an enzymatic complex of about 100-110 Kdal composed of a DNA-dependent ATPase and of an as yet uncharacterized DNA-binding protein. This complex exhibits a limited "helicase" activity on DNA : DNA partial duplexes which probably accounts for the stimulation of DNA polymerase alpha. The other stimulatory factor is obtained from calf thymus. They are the so-called single-stranded DNA binding proteins (DBP) which have a duplex-destabilizing activity. These proteins appear to be heterogeneous with regard to both physical properties (Mr and pI) and functional characteristics (DNA polymerase alpha stimulation, duplex denaturation). The origin and the biological significance of the different molecular forms are discussed.
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PMID:DNA polymerase accessory proteins in mammalian cells. 614 84

We have constructed a map of the genes encoded by a 23,000-nucleotide-pair region of herpes simplex virus type 1. This region, defined by the three adjacent EcoRI fragments N (map coordinates 0.298 to 0.315), F (0.315 to 0.421), and M (0.421 to 0.448), has previously been shown by genetic analysis to contain the genes for thymidine kinase, nucleocapsid protein p40, glycoprotein B, DNA-binding protein, and DNA polymerase. We report the identification and mapping of RNAs defining 13 viral genes encoded by the region 0.298 to 0.448. The transcriptional pattern shows families of overlapping messages, similar to those observed in other regions of the viral genome. We also isolated mutants representing four distinct complementation groups and physically mapped several of the mutations to regions within EcoRI fragment F by marker rescue. Mutations representing complementation groups 1-9 (glycoprotein B), 1-1 (DNA-binding protein), and 1-3 (DNA polymerase) were mapped to coordinates 0.361 to 0.368 to 0.411, and 0.411 to 0.421, respectively. A fourth previously undefined complementation group was mapped to the region between glycoprotein B and DNA-binding protein. Comparing the transcription mapping with marker rescue data suggests that the genes for glycoprotein B, DNA-binding protein, DNA polymerase, and nucleocapsid protein p40 are expressed as 3.3-, 4.2-, 4.3- or 4.2- or both, and 2.4-kilobase mRNAs, respectively.
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PMID:Transcriptional and genetic analyses of the herpes simplex virus type 1 genome: coordinates 0.29 to 0.45. 619 14


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