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)

cDNA encoding the human homologue of mouse APEX nuclease was isolated from a human bone-marrow cDNA library by screening with cDNA for mouse APEX nuclease. The mouse enzyme has been shown to possess four enzymatic activities, i.e., apurinic/apyrimidinic endonuclease, 3'-5' exonuclease, DNA 3'-phosphatase and DNA 3' repair diesterase activities. The cDNA for human APEX nuclease was 1420 nucleotides long, consisting of a 5' terminal untranslated region of 205 nucleotide long, a coding region of 954 nucleotide long encoding 318 amino acid residues, a 3' terminal untranslated region of 261 nucleotide long, and a poly(A) tail. Determination of the N-terminal amino acid sequence of APEX nuclease purified from HeLa cells showed that the mature enzyme lacks the N-terminal methionine. The amino acid sequence of human APEX nuclease has 94% sequence identity with that of mouse APEX nuclease, and shows significant homologies to those of Escherichia coli exonuclease III and Streptococcus pneumoniae ExoA protein. The coding sequence of human APEX nuclease was cloned into the pUC18 SmaI site in the control frame of the lacZ promoter. The construct was introduced into BW2001 (xth-11, nfo-2) strain and BW9109 (delta xth) strain cells of E. coli. The transformed cells expressed a 36.4 kDa polypeptide (the 317 amino acid sequence of APEX nuclease headed by the N-terminal decapeptide derived from the part of pUC18 sequence), and were less sensitive to methylmethanesulfonate and tert-butyl-hydroperoxide than the parent cells. The N-terminal regions of the constructed protein and APEX nuclease were cleaved frequently during the extraction and purification processes of protein to produce the 31, 33 and 35 kDa C-terminal fragments showing priming activities for DNA polymerase on acid-depurinated DNA and bleomycin-damaged DNA. Formation of such enzymatically active fragments of APEX nuclease may be a cause of heterogeneity of purified preparations of mammalian AP endonucleases. Based on analyses of the deduced amino acid sequence and the active fragments of APEX nuclease, it is suggested that the enzyme is organized into two domains, a 6 kDa N-terminal domain having nuclear location signals and 29 kDa C-terminal, catalytic domain.
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PMID:cDNA cloning, sequencing, expression and possible domain structure of human APEX nuclease homologous to Escherichia coli exonuclease III. 162 44

Monoacetyl-hydroxyaminoquinoline 1-oxide (Ac-HAQO) is a model of the ultimate form of the carcinogen 4-nitroquinoline 1-oxide and so it is useful to characterize its reactions with DNA. We find that Ac-HAQO produces one single-strand break (SSB) for every 60 adducts formed in a reaction with supercoiled DNA. The SSBs do not appear to be formed by a free radical reaction and they are distributed throughout the DNA molecule without regard to nucleotide specificity. Unique DNA fragments were reacted with Ac-HAQO. These substrates could not be degraded by the 3'-5' exonuclease action of T4 DNA polymerase unless they were first cleaved by a restriction endonuclease. This indicated that the ends of all the DNA molecules were blocked by adduct formation in spite of the low overall frequency of adducts per DNA molecule.
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PMID:The reaction of acetyl-4-hydroxyaminoquinoline 1-oxide with DNA: quantitation of single-strand break formation and hyper-reactivity of DNA termini. 164 83

Most DNA polymerases are multifunctional proteins that possess both polymerizing and exonucleolytic activities. For Escherichia coli DNA polymerase I and its relatives, polymerase and exonuclease activities reside on distinct, separable domains of the same polypeptide. The catalytic subunits of the alpha-like DNA polymerase family share regions of sequence homology with the 3'-5' exonuclease active site of DNA polymerase I; in certain alpha-like DNA polymerases, these regions of homology have been shown to be important for exonuclease activity. This finding has led to the hypothesis that alpha-like DNA polymerases also contain a distinct 3'-5' exonuclease domain. We have introduced conservative substitutions into a 3'-5' exonuclease active site homology in the gene encoding herpes simplex virus DNA polymerase, an alpha-like polymerase. Two mutants were severely impaired for viral DNA replication and polymerase activity. The mutants were not detectably affected in the ability of the polymerase to interact with its accessory protein, UL42, or to colocalize in infected cell nuclei with the major viral DNA-binding protein, ICP8, suggesting that the mutation did not exert global effects on protein folding. The results raise the possibility that there is a fundamental difference between alpha-like DNA polymerases and E. coli DNA polymerase I, with less distinction between 3'-5' exonuclease and polymerase functions in alpha-like DNA polymerases.
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PMID:Polymerization activity of an alpha-like DNA polymerase requires a conserved 3'-5' exonuclease active site. 165 64

The antiviral distamycin A and its phenyl mustard derivative FCE24517 possessing antitumor activity were tested for their ability to inhibit macromolecular synthesis in three human and one murine cell line. While distamycin A was poorly active in these systems, FCE24517 inhibited DNA synthesis efficiently, RNA synthesis to a lower extent and had little effect on protein synthesis. These findings suggest that the in vivo activity of FCE24517 derives from the specific inhibition of DNA synthesis. When the two drugs were tested on several enzymes involved in human DNA metabolism a strikingly similar pattern of inhibition appeared, with distamycin A being the more potent. Both drugs showed: A), no inhibitory activity against thymidine kinase and DNA primase; B), low activity against DNA topoisomerases I and II and the 3'-5' exonuclease associated with the DNA polymerase epsilon; C), high activity against DNA polymerases alpha and epsilon, uracil-DNA glycosylase and the joining activity of the replicative DNA ligase; D), the highest inhibitory activity against the AMP-dependent DNA relaxing activity of DNA ligase. The strong in vitro inhibition of several DNA enzymatic activities, including DNA ligase, do not match with the in vivo activities of the two drugs. However a unique difference was observed: only FCE24517 inhibited the DNA-independent reaction of adenylation of human DNA ligase while the adenylation reaction of T4 and E. coli DNA ligase was unaffected by either drug. It is still unclear whether these properties are relevant for modulating the killing activity of FCE24517 against proliferating cells both in culture and in vivo. Nevertheless FCE24517 is the first known molecule capable of interacting directly and specifically with human DNA ligase.
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PMID:Specific inhibition of human DNA ligase adenylation by a distamycin derivative possessing antitumor activity. 170 93

The human immunodeficiency virus 1 (HIV-1) reverse transcriptase (RT) is a protein of 66 kDa, p66, which contains two domains, an amino-terminal DNA polymerase and an RNase H at the carboxy terminus of the molecule. In order to characterize the mode of action of the RNase H, two previously described mutant enzymes were used, with substitutions in the highly conserved histidine 539, which was mutated to the neutral amino acid asparagine and to the negatively charged aspartate. The purified wild-type (wt) and mutant (mt) enzyme activities are analyzed here using RNA-DNA hybrids consisting of in vitro transcribed RNA that harbors the polypurine tract (PPT) from HIV-1 and DNA oligonucleotides complementary to the PPT or to other regions of the RNA. Analysis of the radioactively labeled RNA of these model hybrids after RNase H treatment indicates that both, wt and mt enzymes, are capable of cleaving the RNA in an endonucleolytic manner. The mt enzymes exhibit a severely reduced exonuclease activity. They are more sensitive towards salt and competition with excess of unlabeled hybrid, suggesting a reduced substrate binding affinity. DNA elongation by the RT is coupled with RNA hydrolysis by the 3'-5' exonuclease of the wt RNase H. The RNase Hmt of the mt enzymes, however, does not exhibit such processive 3'-5' exonuclease activity during DNA synthesis but gives rise to sporadic endonucleolytic cuts, whereas the RT is not affected. The endonuclease activities of the RNase H mt enzymes exhibit cleavage preferences in the absence or presence of DNA synthesis different from those of the wt enzyme. They cannot recognize specific sequences required to generate a PPT-primer and therefore cannot initiate plus-strand DNA synthesis in vitro at the 3' end of the PPT, which is essential for viral replication.
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PMID:Mutations of a conserved residue within HIV-1 ribonuclease H affect its exo- and endonuclease activities. 171 5

Monoacetyl-4-hydroxyaminoquinoline 1-oxide (Ac-HAQO) reacts with DNA to form adducts at the C8- and N2-positions of guanine and with the N6-position of adenine. Only the N2-guanine adduct blocks the 3'-5' exonuclease action of phage T4 DNA polymerase. Piperidine treatment cleaves the DNA at sites bearing C8-guanine adducts. The N2-position of guanine lies in the minor groove of DNA, whereas the C8-position of guanine occupies the major groove. We have taken advantage of these characteristics to employ Ac-HAQO in conjunction with either T4 DNA polymerase or piperidine in a footprinting technique to probe the interaction of the Escherichia coli integration host factor (IHF) with its binding site. We show that when IHF binds to its recognition site both the N2- and C8-positions of guanines are protected from modification by AcHAQO. In addition, the binding of IHF to DNA was prevented when either an N2- or a C8-AQO adduct was present in the binding site. When dimethylsulfate was used as the footprinting reagent, IHF protected against methylation of the N3 position of adenine in the minor groove but not the N7 position of guanine in the major groove. The difference in results obtained with the two reagents is ascribed to their relative sizes. Both DMS and AcHAQO are excluded by IHF from the minor groove, but only the larger AcHAQO molecule is excluded from the major groove.
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PMID:Use of monoacetyl-4-hydroxyaminoquinoline 1-oxide to probe contacts between guanines and protein in the minor and major grooves of DNA. Interaction of Escherichia coli integration host factor with its recognition site in the early promoter and transposition enhancer of bacteriophage Mu. 183 56

Understanding the response of DNA polymerase to the encountered damage in a template is a key to assessing lethal and mutagenic events of cells exposed to genotoxic agents. In the present study M13 (or f1) DNA templates containing 4 types of thymine damages were prepared, and DNA synthesis was carried out in vitro with the templates. The extent of inhibition of DNA synthesis by the damages was evaluated by measuring [3H]dTMP incorporation. Furthermore, newly synthesized DNA was analyzed on a sequencing gel to determine termination sites of DNA synthesis. The results showed that DNA synthesis was differentially inhibited by the damages, and the termination sites of DNA synthesis were dependent on the structures of the damages and the 3'-5' exonuclease activity of DNA polymerase used.
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PMID:Effects of base damages on DNA replication. 184 66

9-(2-Phosphonylmethoxyethyl)adenine (PMEA) is a new antiviral compound with activity against herpes simplex virus (HSV) and retroviruses including human immunodeficiency virus. Although it has been suggested that the anti-HSV action of PMEA is through inhibition of the viral DNA polymerase via the diphosphorylated metabolite of PMEA (PMEApp), no conclusive evidence for this has been presented. We report that in cross-resistance studies, a PMEA-resistant HSV variant (PMEAr-1) was resistant to phosphonoformic acid, a compound which directly inhibits the HSV DNA polymerase. In addition, phosphonoformic acid-resistant HSV variants with defined drug resistance mutations within the HSV DNA polymerase gene were resistant to PMEA. Furthermore, the HSV DNA polymerase purified from PMEAr-1 was resistant to PMEApp in comparison with the enzyme from the parental virus. Moreover, PMEA inhibited HSV DNA synthesis in cell culture. These results provide strong evidence that HSV DNA polymerase is the major target for the anti-viral action of PMEA. Further studies showed that HSV DNA polymerase incorporated PMEApp into DNA in vitro, while the HSV polymerase-associated 3'-5' exonuclease was able to remove the incorporated PMEA. Thus, the inhibition of HSV DNA polymerase by PMEApp appears to involve chain termination after its incorporation into DNA.
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PMID:Herpes simplex virus-specified DNA polymerase is the target for the antiviral action of 9-(2-phosphonylmethoxyethyl)adenine. 184 64

The kinetic parameters governing incorporation of correct and incorrect bases into synthetic DNA duplexes have been investigated for Escherichia coli DNA polymerase I [Klenow fragment (KF)] and for two mutants, Tyr766Ser and Tyr766Phe. Tyr766 is located at the C-terminus of helix O in the DNA-binding cleft of KF. The catalytic efficiency for correct incorporation of dNTP is reduced 5-fold for Tyr766Ser. The catalytic efficiencies of all 12 possible misincorporations have been determined for both KF and Tyr766Ser by using single-turnover kinetic conditions and a form of the enzyme that is devoid of the 3'-5' exonuclease activity because of other single amino acid replacements. Tyr766Ser displays an increased efficiency of misincorporation (a reduction in fidelity) for several of the 12 mismatches. The largest increase in efficiency of misincorporation for Tyr766Ser occurs for the misincorporation of TMP opposite template guanosine, a 44-fold increase. In contrast, the efficiencies of misincorporation of dAMP opposite template A, G, or C are little affected by the mutation. A determination of the kinetic parameters associated with a complete kinetic scheme has been made for Tyr766Ser. The rate of addition of the next correct nucleotide onto a preexisting mismatch is decreased for Tyr766Ser. The fidelity of Tyr766Phe was not substantially different from that of KF for the misincorporations examined, indicating that it is the loss of the phenolic ring of the side chain of Tyr766 that leads to the significant decrease in fidelity. The results indicate that KF actively participates in the reduction of misincorporations during the polymerization event and that Tyr766 plays an important role in maintaining the high fidelity of replication by KF.
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PMID:A mutant of DNA polymerase I (Klenow fragment) with reduced fidelity. 189 34

It is speculated that DNA polymerases which duplicate chromosomes are dimeric to provide concurrent replication of both leading and lagging strands. DNA polymerase III holoenzyme (holoenzyme), is the 10-subunit replicase of the Escherichia coli chromosome. A complex of the alpha (DNA polymerase) and epsilon (3'-5' exonuclease) subunits of the holoenzyme contains only one of each protein. Presumably, one of the eight other subunit(s) functions to dimerize the alpha epsilon polymerase within the holoenzyme. Based on dimeric subassemblies of the holoenzyme, two subunits have been elected as possible agents of polymerase dimerization, one of which is the tau subunit (McHenry, C. S. (1982) J. Biol. Chem. 257, 2657-2663). Here, we have used pure alpha, epsilon, and tau subunits in binding studies to determine whether tau can dimerize the polymerase. We find tau binds directly to alpha. Whereas alpha is monomeric, tau is a dimer in its native state and thereby serves as an efficient scaffold to dimerize the polymerase. The epsilon subunit does not associate directly with tau but becomes dimerized in the alpha epsilon tau complex by virtue of its interaction with alpha. We have analyzed the dimeric alpha epsilon tau complex by different physical methods to increase the confidence that this complex truly contains a dimeric polymerase. The tau subunit is comprised of the NH2-terminal two-thirds of tau but does not bind to alpha epsilon, identifying the COOH-terminal region of tau as essential to its polymerase dimerization function. The significance of these results with respect to the organization of subunits within the holoenzyme is discussed.
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PMID:Constitution of the twin polymerase of DNA polymerase III holoenzyme. 191 87


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