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)

Recently we reported that a DNA fragment, GCCAAAGC, forms an extraordinarily stable hairpin structure with two G-C pairs at the terminus and A-A-A stacked structure. The sequence is present at the replication origin of bacteriophage G4 ssDNA, and so on. Several kinds of possible hairpin structures, corresponding to the replication origin of phage G4, were synthesized and their secondary structures were examined. It was found that the fragments are able to form interconvertible hairpin structures depending on the length of the base-paired regions. The hairpin structure consisting of GCGAAAGC was not digested by the exonuclease activity of T4 DNA polymerase and it was stable enough to be only minimally bound by a single-stranded DNA binding protein.
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PMID:Unique hairpin structures occurring at the replication origin of phage G4 DNA. 222 81

Characterization of the domain structure of DNA polymerase beta is reported. Large scale overproduction of the rat protein in Escherichia coli was achieved, and the purified recombinant protein was verified by sequencing tryptic peptides. This protein is both a single-stranded DNA binding protein and a DNA polymerase consisting of one polypeptide chain of 334 amino acids. As revealed by controlled proteolysis experiments, the protein is organized in two relatively protease-resistant segments linked by a short protease-sensitive region. One of these protease-resistant segments represents the NH2-terminal 20% of the protein. This NH2-terminal domain (of about 75 residues) has strong affinity for single-stranded nucleic acids. The other protease-resistant segment, representing the COOH-terminal domain of approximately 250 residues, does not bind to nucleic acids. Neither domain, tested as purified proteins, has substantial DNA polymerase activity. The results suggest that the NH2-terminal domain is principally responsible for the template binding activity of the intact protein.
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PMID:Studies of the domain structure of mammalian DNA polymerase beta. Identification of a discrete template binding domain. 240 80

The most abundant single-stranded DNA binding protein (SSB) found in ovaries of the frog, Xenopus laevis, was purified to electrophoretic homogeneity. Under physiological conditions, the purified SSB lowered the Tm of poly[d(A-T)] and stimulated DNA synthesis by the homologous DNA polymerase DNA primase alpha complex on single-stranded DNA templates. These properties are characteristic of a bona fide single-stranded DNA binding protein. The Stokes radius of native SSB was calculated to be 45 A, corresponding to a molecular mass of about 140 kDa. On SDS polyacrylamide gels, the SSB migrated as a single band with a molecular mass of 36 kDa. We assumed, therefore, that the SSB was a tetramer of 36 kDa subunits. We subsequently discovered that the SSB was LDH, D-lactate dehydrogenase, EC 1.1.1.28. Purified SSB has high LDH specific activity. Following electrophoresis on SDS polyacrylamide gels, the 36 kDa subunits were renatured and exhibited LDH activity. The amino-acid composition of X. laevis SSB/LDH was similar to that of LDH from other species and to other reported single-stranded DNA binding proteins. Mammalian SSB/LDH also preferentially bound single-stranded DNA. Mammalian SSB/LDH bound to RNA as demonstrated by affinity chromatography on poly(A)-agarose and by its effect on translation of mRNA in vitro.
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PMID:A major single-stranded DNA binding protein from ovaries of the frog, Xenopus laevis, is lactate dehydrogenase. 247 Apr 7

Mechanisms that could operate to initiate pBR322 DNA replication in the absence of RNase H and DNA polymerase I are described. Two different pathways leading to extensive unwinding of pBR322 DNA have been observed under DNA replication reaction conditions in vitro. In the presence of RNA polymerase and DNA gyrase, specifically initiated RNA II (the leading-strand primer precursor) can form an RNA-DNA hybrid with the template that starts just upstream of the origin of DNA replication and continues for about 3 kilobases. Subsequent digestion of the RNA in this RNA-pBR322 DNA hybrid results in the formation of a highly unwound DNA termed form I. If DNA gyrase is absent during the RNA polymerase-catalyzed elongation of RNA II, a stable RNA-pBR322 DNA hybrid can still form that is localized to the origin region of the genome. Formation of this hybrid activates the primosome assembly site present on the lagging-strand DNA template, by displacing it to a single-stranded conformation, thereby allowing preprimosome assembly. Once assembled, the DNA helicase activity of the preprimosome, in the presence of the single-stranded DNA binding protein and DNA gyrase but in the absence of any further transcription, can also result in extensive unwinding of pBR322 DNA. The product of this reaction, form I DNA, is more unwound than form I DNA. The formation of both form I and form I DNA is inhibited by the presence of excess RNA I, as well as by RNase H at concentrations sufficient to catalyze the normal processing of RNA II required for initiation of leading-strand DNA synthesis. These results suggest that RNA II-pBR322 DNA hybrid formation is essential to permit preprimosome assembly during pBR322 DNA replication under conditions where both RNase H and DNA polymerase I are absent.
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PMID:Transcriptional activation of pBR322 DNA can lead to duplex DNA unwinding catalyzed by the Escherichia coli preprimosome. 247 95

Seven herpes simplex virus (HSV) genes have been shown recently to be necessary and sufficient to support the replication of origin-containing plasmids. Two of these genes (pol and dbp) encode well-known DNA replication proteins (the DNA polymerase and the major single-stranded DNA binding protein), and a third gene (UL42) encodes a previously identified infected-cell protein which binds tightly to double-stranded DNA. The products of the four remaining genes have not previously been identified. Using the predicted amino acid sequence data (D.J. McGeoch, M.A. Dalrymple, A. Dolan, D. McNab, L.J. Perry, P. Taylor, and M.D. Challberg, J. Virol. 62:444-453; D.J. McGeoch and J.P. Quinn, Nucleic Acids Res. 13:8143-8163), we have raised rabbit antisera against the products of all seven genes. We report here the use of these reagents to identify these proteins in infected cells. All seven proteins localized to the nucleus and were expressed in a manner consistent with the idea that they are the products of early genes. Various immunological assays suggest that four of these proteins (UL5, UL8, UL9, and UL52) are made in infected cells in very low abundance relative to the other three. To improve our ability to study these proteins, we have expressed UL5, UL8, UL9, and UL52 in insect cells by using the baculovirus expression system. The HSV protein made in insect cells were immunoprecipitable with the appropriate antisera, and the size of each protein was indistinguishable from the size of the corresponding protein made in HSV-infected Vero cells. Our data offer strong support for the accuracy of open reading frames proposed by McGeoch et al. In addition, the antisera and the overproduced HSV replication proteins should be useful reagents with which to analyze the biochemistry of HSV DNA replication.
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PMID:Herpes simplex virus type 1 gene products required for DNA replication: identification and overexpression. 253 26

By using an in vitro system for R1 plasmid replication dependent on a plasmid-encoded repA protein and host dnaA protein, 5' ends of the nascent leading strand were located at positions 1986-1992, some 380 base pair downstream of oriR. Analyses of early replication intermediates generated in vitro in the presence of dideoxy TTP also indicated that replication initiates about 400 base pair downstream of oriR and proceeds unidirectionally. When a 418-base single-stranded DNA from position 1778 to 2195, derived from the leading strand template, was cloned onto an M13 vector, the chimeric single-stranded phage could be replicated in vitro with only single-stranded DNA binding protein, primase (dnaG gene product), and DNA polymerase III holoenzyme. Furthermore, the priming occurred at a site identical to leading strand initiation. These results strongly suggest that the leading strand synthesis is primed by primase alone. The lagging strand synthesis is specifically terminated at position 1515 or 1516 within oriR, preventing further leftward fork movement. Based on these results, a scheme of R1 plasmid replication is presented.
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PMID:Leading strand synthesis of R1 plasmid replication in vitro is primed by primase alone at a specific site downstream of oriR. 254 61

The primosome is a mobile multiprotein DNA replication-priming apparatus that requires seven Escherichia coli proteins (replication factor Y (protein n'), proteins n and n", and the products of the dnaB, dnaC, dnaT, and dnaG genes) for assembly at a specific site (termed a primosome assembly site) on single-stranded DNA binding protein-coated single-stranded DNA. Two of the protein components of the primosome have intrinsic DNA helicase activity. The DNA B protein acts in the 5'----3' direction, whereas factor Y acts in the 3'----5' direction. The primosome complex has DNA helicase activity when present at a replication fork in conjunction with the DNA polymerase III holoenzyme. In this report, evidence is presented that the multiprotein primosome per se can act as a DNA helicase in the absence of the DNA polymerase III holoenzyme. The primosome DNA helicase activity can be manifested in either direction along the DNA strand. The directionality of the primosome DNA helicase activity is modulated by the concentration and type of nucleoside triphosphate present in the reaction mixture. This DNA helicase activity requires all the preprimosomal proteins (the primosomal proteins minus the dnaG-encoded primase). Preprimosome complexes must assemble at a primosome assembly site in order to be loaded onto the single-stranded DNA and act subsequently as a DNA helicase. The 5'----3' primosome DNA helicase activity requires a 3' single-stranded tail on the fragment to be displaced, while the 3'----5' activity does not require a 5' single-stranded tail on the fragment to be displaced. Multienzyme preprimosomes moving in either direction are capable of associating with the primase to form complete primosomes that can synthesize RNA primers.
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PMID:The Escherichia coli primosome can translocate actively in either direction along a DNA strand. 254 99

The nearly homogeneous 9 S DNA polymerase alpha from calf thymus contains a primase activity that allows priming of DNA synthesis on single-stranded templates in the presence of ribonucleoside triphosphates. Both on synthetic and natural single-stranded templates, RNA primers of 8-15 nucleotides in length are formed. In the absence of dNTPs, primers of some hundred nucleotides in length are observable. ATP and/or GTP are required for the priming reaction. UTP and CTP cannot initiate the RNA synthesis. M13 single-stranded DNA can be converted to the nicked double helical form upon primase-primed replication by the 9 S enzyme. Priming occurs mostly at specific sites on the M13 genome and replication products of up to 6000 nucleotides in length are formed. In the presence of the single-stranded DNA binding protein from Escherichia coli, specificity of priming is strongly increased. The primase is inhibited by salt and actinomycin; it is insensitive to alpha-amanitin and N-ethylmaleimide. Due to the strong complex formation between DNA polymerase and primase, it has not been possible to separate the two activities of the multisubunit 9 S enzyme.
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PMID:The primase activity of DNA polymerase alpha from calf thymus. 257 65

We have purified and characterized a single-stranded DNA binding protein (N4 SSB) induced after coliphage N4 infection. It has a monomeric molecular weight of 31,000 and contains 10 tyrosine and 1-2 tryptophan amino acid residues. Its fluorescence spectrum is dominated by the tyrosine residues, and their fluorescence is quenched when the protein binds single-stranded DNA. Fluorescence quenching was used as an assay to quantitate binding of the protein to single-stranded nucleotides. The N4 single-stranded DNA binding protein binds cooperatively to single-stranded nucleic acids and binds single-stranded DNA more tightly than RNA. The binding involves displacement of cations from the DNA and anions from the protein. The apparent binding affinity is very salt-dependent, decreasing as much as 1,000-fold for a 10-fold increase in NaCl concentration. The degree of cooperativity (omega) is relatively independent of salt concentration. At 37 degrees C in 0.22 M NaCl, the protein has an intrinsic binding constant for M13 viral DNA of 3.8 x 10(4) M-1, a cooperativity factor omega of 300, and binding site size of 11 nucleotides per monomer. The protein lowers the melting point of poly(dA.dT).poly(dA-dT) by greater than 60 degrees C but cannot lower the melting transition or assist in the renaturation of natural DNA. N4 single-stranded DNA binding protein enhances the rate of DNA synthesis catalyzed by the N4 DNA polymerase by increasing the processivity of the N4 DNA polymerase and melting out hairpin structures that block polymerization.
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PMID:Purification and characterization of the coliphage N4-coded single-stranded DNA binding protein. 266 66

A DNA replication system was developed that could generate rolling-circle DNA molecules in vitro in amounts that permitted kinetic analyses of the movement of the replication forks. Two artificial primer-template DNA substrates were used to study DNA synthesis catalyzed by the DNA polymerase III holoenzyme in the presence of either the preprimosomal proteins (the primosomal proteins minus the DNA G primase) and the Escherichia coli single-stranded DNA binding protein or the DNA B helicase alone. Helicase activities have recently been demonstrated to be associated with the primosome, a mobile multiprotein priming apparatus that requires seven E. coli proteins (replication factor Y (protein n'), proteins n and n'', and the products of the dnaB, dnaC, dnaG, and dnaT genes) for assembly, and with the DNA B protein. Consistent with a rolling-circle mechanism in which a helicase activity permitted extensive (-) strand DNA synthesis on a (+) single-stranded, circular DNA template, the major DNA products formed were multigenome-length, single-stranded, linear molecules. The replication forks assembled with either the preprimosome or the DNA B helicase moved at the same rate (approximately 730 nucleotides/s) at 30 degrees C and possessed apparent processivities in the range of 50,000-150,000 nucleotides. The single-stranded DNA binding protein was not required to maintain this high rate of movement in the case of leading strand DNA synthesis catalyzed by the DNA polymerase III holoenzyme and the DNA B helicase.
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PMID:The Escherichia coli preprimosome and DNA B helicase can form replication forks that move at the same rate. 282 2


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