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 Km and vmax values for oligothymidylates d(pT)2-16 in reaction of 3'-5'-exonuclease hydrolysis catalyzed by Klenow fragment were measured in the absence and presence of poly(dA) template without the poly(dA), the Km values for oligonucleotides are slightly dependent on their length. The rate of oligothymidylates hydrolysis increases with their length and for d(pT)16 it is about 190-times higher than for d(pT)2. The addition on poly(dA) does not lead to an essential change of the Km values for d(pT)2-16, but raises the rate of d(pT)2-7 hydrolysis 2-17-fold and at the same time lowers the efficiency of d(pT)8-16 hydrolysis. The Km values for d(pC)10, d(pA)19 and d(pT)10 are nearly the same. However the velocity of d(pC)10 hydrolysis is approximately 1,2 and 7,8-times higher than for d(pA)10 and d(pC)10, respectively d(pC)10, d(pA)10 and d(pT)10 under conditions of interaction with the template-binding site raise the rate of hydrolysis of d(pT)2 combined with the exonuclease center, with various efficiency. Under similar conditions, d(pT)8, d(pT)10 and d(pT)16 as templates activated hydrolysis of d(pT)2. The dependence of the Klenow fragment exonuclease activity both on the length and structure of the template and on the length of the hydrolyzed oligonucleotide was suggested.
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PMID:[Dependence of 3'-5-exonuclease activity of a fragment of Klenow DNA polymerase I from Escherichia coli on the length and structure of the cleaved oligonucleotide]. 196 5

AMP and NaF each taken separately were shown to activate DNA polymerization catalyzed by Klenow fragment of DNA polymerase I by means of interaction of AMP or NaF with 3'----5'-exonuclease center of the enzyme. In the presence of NaF which is a selective inhibitor of 3'----5'-exonuclease center, AMP is an inhibitor of polymerization competitive with respect to dATP. Ki values and the pattern of inhibition with respect to dATP were determined for AMP, ADP, ATP, carboxymethylphosphonyl-5'-AMP, Pi, PPi, PPPi, methylenediphosphonic acid and its ethylated esters, phosphonoformic acid, phosphonoacetic acid and its ethylated esters as well as for some bicarbonic acids in the reactions of DNA polymerization catalyzed by Klenow fragment of DNA polymerase I (in the presence of NaF) and DNA polymerase alpha from human placenta in the presence of poly(dT) template and r(pA)10 primer. All nucleotides and their analogs were found to be capable of competing with dATP for the active center of the enzyme. Most of the analogs of PPi and phosphonoacetic acid are inhibitors of Klenow fragment competitive with respect to dATP. Nowever these analogs display a mixed-type inhibition in the case of human DNA polymerase alpha. We postulated a similar mechanism of interaction for dNTP with both DNA-polymerases. It is suggested that each phosphate group of PPi makes equal contribution to the interaction with DNA polymerases and that the distance between the phosphate groups is important for this interaction. beta-phosphate of NTP or dNTP is suggested to make negligible contribution to the efficiency of the formation of enzyme complexes with dNTP. beta-phosphate is likely to be an essential point of PPi interaction with the active center of proteins during the cleavage of the alpha-beta-phosphodiester bond of dNTP in the reaction of DNA polymerization.
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PMID:[Interaction of dNTP-binding sites of human DNA polymerase alpha and The Klenow fragment of Escherichia coli DNA polymerase I with nucleotides, pyrophosphate and their analogs]. 216 89

[3H]dGMP-3'-labelled, activated salmon testis DNA and [32P]dGMP-5'-labelled open circular M13 DNA were reacted with cis-diamminedichloroplatinum(II), cis-diamminechloroaquaplatinum(II), cis-diamminediaquaplatinum(II) or trans-diamminechloroaquaplatinum(II). The reaction was arrested after arbitrary times by adjustment to slightly alkaline solution conditions. The platinum-containing DNA was digested with Escherichia coli DNA polymerase I. The progress of nucleotide release was measured by acid precipitation of undigested DNA. Solubilized nucleotides and adducts were analyzed by HPLC. The 3'-5'-exonuclease activity liberated single-coordinated dGMP-platinum(II) adducts from both cis- and trans-platinum(II) treated salmon testis DNA and a small fraction of adducts of cis-platinum(II) that coordinated two molecules of dGMP. The bisadduct was derived from non-neighboring guanine residues probably located at or close to 3'-termini. This nuclease activity neither cut between nor after neighboring guanine residues crosslinked by cis-platinum(II). No bisadduct was liberated for trans-platinum(II). The 5'-3'-exonuclease activity did not liberate any nucleotide adducts from cis-platinum(II)-treated DNa. However, it removed single-coordinated guanine adducts of trans-diamminedichloroplatinum(II). From the kinetics of the appearance of dGMP monoadducts and the inhibition of digestion, a reaction scheme is formulated for the reaction of platinum(II) complexes with DNA that confirms and extends the previously published one [W. Schaller, H. Reisner & E. Holler (1987) Biochemistry 26, 943-950]. The longevity of the dGMP monoadduct intermediate is discussed in the context of the efficiency of cis-diamminedichloroplatinum(II) as an antitumor drug.
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PMID:Escherichia coli DNA polymerase I: inherent exonuclease activities differentiate between monofunctional and bifunctional adducts of DNA and cis- or trans-diamminedichloroplatinum(II). An exonuclease investigation of the kinetics of the adduct formation. 216 53

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

Five proteins purified from mouse cells acting together efficiently convert a single-stranded circular DNA template to covalently closed duplex circle by a discontinuous mechanism. DNA polymerase alpha/primase with the assistance of alpha accessory factor covers the single-stranded circle with RNA-primed DNA fragments. Primers are removed by a combination of RNase H-1 and a 5'-exonuclease that was identified by its ability to complete this in vitro system. The 5'-exonuclease is required to remove residual one or two ribonucleotides at the primer/DNA junction that are resistant to RNase H-1. Gap filling is by the DNA polymerase alpha/primase, and DNA ligase I converts the DNA fragments to continuous strand. The concerted action of the five proteins emulates synthesis of the staging strand at the replication fork.
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PMID:Discontinuous DNA synthesis by purified mammalian proteins. 217 Apr 12

The 3',5'-exonuclease center of the Klenow fragment of E. coli DNA polymerase I (FK) was selectively blocked by NaF. The latter was shown to forbid the binding of nucleotides and their analogs to the enzyme exonuclease center. In the presence of poly(dT).r(pA)10 template.primer complex and NaF, we observed AMP, ADP, ATP, PPi and dATP to be competitive inhibitors of the FK-catalyzed DNA polymerization. The interactions of the nucleotides with FK and human DNA polymerase alpha were compared to reveal similarity of binding to the DNA polymerizing centers. Structural components of dNTP and PPi playing key roles in forming complexes with pro- and eukaryotic DNA polymerases were identified.
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PMID:Interaction of dNTP, pyrophosphate and their analogs with the dNTP-binding sites of E. coli DNA polymerase I Klenow fragment and human DNA polymerase alpha. 217 14

DNA polymerase I (Pol I) is an enzyme of DNA replication and repair containing three active sites, each requiring divalent metal ions such as Mg2+ or Mn2+ for activity. As determined by EPR and by 1/T1 measurements of water protons, whole Pol I binds Mn2+ at one tight site (KD = 2.5 microM) and approximately 20 weak sites (KD = 600 microM). All bound metal ions retain one or more water ligands as reflected in enhanced paramagnetic effects of Mn2+ on 1/T1 of water protons. The cloned large fragment of Pol I, which lacks the 5',3'-exonuclease domain, retains the tight metal binding site with little or no change in its affinity for Mn2+, but has lost approximately 12 weak sites (n = 8, KD = 1000 microM). The presence of stoichiometric TMP creates a second tight Mn2+ binding site or tightens a weak site 100-fold. dGTP together with TMP creates a third tight Mn2+ binding site or tightens a weak site 166-fold. The D424A (the Asp424 to Ala) 3',5'-exonuclease deficient mutant of the large fragment retains a weakened tight site (KD = 68 microM) and has lost one weak site (n = 7, KD = 3500 microM) in comparison with the wild-type large fragment, and no effect of TMP on metal binding is detected. The D355A, E357A (the Asp355 to Ala, Glu357 to Ala double mutant of the large fragment of Pol I) 3',5'-exonuclease-deficient double mutant has lost the tight metal binding site and four weak metal binding sites. The binding of dGTP to the polymerase active site of the D355A,E357A double mutant creates one tight Mn2+ binding site with a dissociation constant (KD = 3.6 microM), comparable with that found on the wild-type enzyme, which retains one fast exchanging water ligand. Mg2+ competes at this site with a KD of 100 microM. It is concluded that the single tightly bound Mn2+ on Pol I and a weakly bound Mn2+ which is tightened 100-fold by TMP are at the 3',5'-exonuclease active site and are essential for 3',5'-exonuclease activity, but not for polymerase activity. Additional weak Mn2+ binding sites are detected on the 3',5'-exonuclease domain, which may be activating, and on the polymerase domain, which may be inhibitory. The essential divalent metal activator of the polymerase reaction requires the presence of the dNTP substrate for tight metal binding indicating that the bound substrate coordinates the metal.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Metal binding to DNA polymerase I, its large fragment, and two 3',5'-exonuclease mutants of the large fragment. 220 84

A complex network of interacting proteins and enzymes is required for DNA replication. Much of our present understanding is derived from studies of the bacterium Escherichia coli and its bacteriophages T4 and T7. These results served as a guideline for the search and the purification of analogous proteins in eukaryotes. model systems for replication, such as the simian virus 40 DNA, lead the way. Generally, DNA replication follows a multistep enzymatic pathway. Separation of the double-helical DNA is performed by DNA helicases. Synthesis of the two daughter strands is conducted by two different DNA polymerases: the leading strand is replicated continuously by DNA polymerase delta and the lagging strand discontinuously in small pieces by DNA polymerase alpha. The latter is complexed to DNA primase, an enzyme in charge of frequent RNA primer syntheses on the lagging strand. Both DNA polymerases require several auxiliary proteins. They appear to make the DNA polymerases processive and to coordinate their functional tasks at the replication fork. 3'----5'-exonuclease, mostly part of the DNA polymerase delta polypeptide, can perform proof-reading by excising incorrectly base-paired nucleotides. The short DNA pieces of the lagging strand, called Okazaki fragments, are processed to a long DNA chain by the combined action of RNase H and 5'----3'-exonuclease, removing the RNA primers, DNA polymerase alpha or beta, filling the gap, and DNA ligase, sealing DNA pieces by phosphodiester bond formation. Torsional stress during DNA replication is released by DNA topoisomerases. In contrast to prokaryotes, DNA replication in eukaryotes not only has to create two identical daughter strands but also must conserve higher-order structures like chromatin.
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PMID:Eukaryotic DNA replication. Enzymes and proteins acting at the fork. 226 94

DNA polymerase delta was isolated from human placenta and identified as such on the basis of its association with a 3'- to 5'-exonuclease activity. The association of the polymerase and exonuclease activities was maintained throughout purification and attempted separations by physical or electrophoretic methods. Moreover, ratios of the two activities remained constant during the purification steps, and both activities were inhibited by aphidicolin, oxidized glutathione, and N-ethylmaleimide. The purified enzyme had an estimated molecular weight of 172,000, on the basis of a Stokes radius of 53.6 A and a sedimentation coefficient of 7.8 S. On sodium dodecyl sulfate (SDS) gel electrophoresis, polymerase delta preparations contained a band of ca. 170 kilodaltons (kDa) as well as several smaller polypeptides. The 170-kDa polypeptide was identified as the largest polypeptide component in the preparation possessing DNA polymerase activity by an activity staining procedure following gel electrophoresis in the presence of SDS. Western blotting of DNA polymerase delta with polyclonal antisera also revealed a single 170-kDa immunoreactive polypeptide. Monoclonal antibodies to KB cell polymerase alpha inhibited placental polymerase alpha but did not inhibit DNA polymerase delta, while the murine polyclonal antisera to polymerase delta inhibited delta but not alpha. These findings establish the existence of DNA polymerase delta in a human tissue and support the view that both its polymerase and its exonuclease activities may be associated with a single protein.
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PMID:Human placental DNA polymerase delta: identification of a 170-kilodalton polypeptide by activity staining and immunoblotting. 243 59

The DNA sequence of the polA gene of Streptococcus pneumoniae was determined, and the DNA polymerase I encoded by the gene was purified to homogeneity. Determination of the amino-terminal amino acid sequence of the protein showed it to correspond to the Mr 99,487 polypeptide predicted from the nucleotide sequence. The mRNA transcript was mapped with respect to its sites of initiation and termination in the DNA. Inasmuch as the mRNA begins only two nucleotides before the first codon, it lacks a typical ribosome binding site. Nevertheless, 500 molecules of the protein are produced per cell. Like the Escherichia coli DNA polymerase I, the protein from S. pneumoniae has 5'- and 3'-exonuclease as well as polymerase activities, and it also undergoes a single cleavage on mild proteolysis. Alignment of the two different polymerase I proteins shows 40% of their amino acid residues to be identical. Homology is evident also with the DNA polymerase encoded by phage T7 gene 5. In addition, the amino-terminal regions of the bacterial polymerase I proteins are homologous to the separate 5'-exonuclease protein encoded by phage T7 gene 6. Analysis of the patterns of homology suggests that the bacterial polymerase I may represent the accretion of at least six separate genetic regions.
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PMID:Characterization of the polA gene of Streptococcus pneumoniae and comparison of the DNA polymerase I it encodes to homologous enzymes from Escherichia coli and phage T7. 253 9


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