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)

Several thiosemicarbazone-metal complexes inhibit the RNA dependent DNA polymerase and the transforming ability of Rous sarcoma virus. Some complexes are equally as active as the free ligand whereas the activity of others is greatly enhanced. The 2-formyl pyridine thiosemicarbazone copper (II) complex is the most potent compound of this class that we tested. Some copper complexes of salicylaldehyde derivatives are very active also, particularly N-n-butyl, N-n-hexyl and N-benzylsalicylaldimine; no nickel complex of any salicylaldehyde compound is active. In addition, other metal ligands, such as dithizone, diacetyl bis (mercaptoethylimine), N-butyl thiocarbamate, 0,0' dimethyl dithiophosphate, potassium dithiooxalate, and cis-PtII(NH3)2Cl2 were tested with varying results.
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PMID:Inhibition of the RNA dependent DNA polymerase and the malignant transforming ability of Rous sarcoma virus by thiosemicarbazone-transition metal complexes. 7 67

A series of site-specifically plantinated, covalently closed circular M13 genomes (7250 bp) was constructed in order to evaluate the consequences of DNA template damage induced by the anticancer drug cis-diamminedichloroplatinum(II) (cis-DDP). Here are reported the synthesis and characterization of genomes containing the intrastrand cross-linked adducts cis-[Pt(NH3)2[d(ApG)-N7(1),-N7(2)]], cis-[Pt-(NH3)2[d(GpCpG)-N7(1),-N7(3)]], and trans-[Pt(NH3)2[d(CpGpCpG)-N3(1),-N7(4)]]. These constructs, as well as the previously reported M13 genome containing a site-specifically placed cis-[Pt(NH3)2[d-(GpG)-N7(1),-N7(2)]] adduct, were used to study replication in vitro. DNA synthesis was initiated from a position approximately 177 nucleotides 3' to the individual adducts, and was terminated either by the adducts or by the end of the template, located approximately 25 nucleotides on the 5' side of the adducts. Analysis of the products of these reactions by gel electrophoresis revealed that, on average, bypass of the cis-DDP adducts occurred approximately 10% of the time and that the cis-[Pt(NH3)2[d(GpG)-N7(1),-N7(2)]] intrastrand cross-link is the most inhibitory lesion. The cis-[Pt(NH3)2[(GpCpG)-N7(1),-N7(3)]] adduct allowed a higher frequency of such translesion synthesis (ca. 25%) for two of the polymerases studied, modified bacteriophage T7 polymerase and Escherichia coli DNA polymerase I (Klenow fragment). These enzymes have either low (Klenow) or no (T7) associated 3' to 5' exonuclease activity. Bacteriophage T4 DNA polymerase, which has a very active 3' to 5' exonuclease, was the most strongly inhibited by all three types of cis-DDP adducts, permitting only 2% translesion synthesis. This enzyme is therefore recommended for replication mapping studies to detect the location of cis-DDP-DNA adducts in a heterologous population. The major replicative enzyme of E. coli, the DNA polymerase III holoenzyme, allowed less than 10% adduct bypass. Postreplication restriction enzyme cleavage studies established that the templates upon which translesion synthesis was observed contained platinum adducts, ruling out the possibility that the observed products were due to a small amount of contamination with unplatinated DNA. The effects on in vitro replication of a recently characterized adduct of trans-DDP [Comess, K. M., Costello, C. E., & Lippard, S. J. (1990) Biochemistry 29, 2102-2110] were also evaluated. This adduct provided a poor block both to DNA polymerases and to restriction enzymes.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Replication inhibition and translesion synthesis on templates containing site-specifically placed cis-diamminedichloroplatinum(II) DNA adducts. 131 53

Cis-diaminedichloroplatinum(II) [cDDP] and three related derivatives Pt(mal)(NH3)2, PtCl2(dach) and Pt(mal) (dach) have been observed to possess cytotoxicity against the growth of P388 lymphocytic leukemia cells. DNA synthesis in P388 cells was inhibited by the agents in a manner which was consistent with their ED50 values for cytotoxicity. When P388 cells were treated with these platinum complexes in vitro at doses which caused more than 80% inhibition of DNA synthesis, no significant inhibition was observed for thymidine, kinase, thymidine monophosphate kinase, carbamoyl phosphate synthetase, or aspartate transcarbamoylase activities. Thus, there was no evidence that these agents inhibited de novo purine, pyrmidine, or deoxynucleotide synthesis. All of the agents did inhibit the nuclear DNA polymerase activity, but the extent of inhibition was 20% or less at doses which caused greater than 70% inhibition of DNA synthesis. Thus, the inhibition of DNA synthesis appeared to be due to cisplatinum(II) drug binding to the DNA bases. This was estimated to be 1 atom of platinum per 1500-3000 DNA base pairs which is consistent with other studies. The platinum complexes with chloro leaving ligands caused considerable DNA strand scission by 24 h at 10 times the ED50 dose, most likely a measure of impending cell death. In contrast, the platinum complexes with malonato leaving ligands did not cause significant strand scission by 24 h at similar doses. They also exhibited a significant delay in the inhibition of DNA synthesis. These data were interpreted as resulting from slower monoadduct to diadduct conversion, but it is not possible to eliminate the possibility of a different mode of interaction with DNA or a different mechanism of cytotoxicity for the malonato compounds.
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PMID:Inhibition of nucleic acid synthesis in P388 lymphocytic leukemia cells in culture by cis-platinum derivatives. 170 16

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

The DNA and protein sequences of single-stranded DNA binding proteins (SSBs) encoded by the plP71a, plP231a, and R64 conjugative plasmids have been determined and compared to Escherichia coli SSB and the SSB encoded by F-plasmid. Although the amino acid sequences of all of these proteins are highly conserved within the NH2-terminal two-thirds of the protein, they diverge in the COOH-terminal third region. A number of amino acid residues which have previously been implicated as being either directly or indirectly involved in DNA binding are conserved in all of these SSBs. These residues include Trp-40, Trp-54, Trp-88, His-55, and Phe-60. On the basis of these sequence comparisons and DNA binding studies, a role for Tyr-70 in DNA binding is suggested for the first time. Although the COOH-terminal third of these proteins diverges more than their NH2-terminal regions, the COOH-terminal five amino acid residues of all five of these proteins are identical. In addition, all of these proteins share the characteristic property of having a protease resistant, NH2-terminal core and an acidic COOH-terminal region. Despite the high degree of sequence homology among the plasmid SSB proteins, the F-plasmid SSB appears unique in that it was the only SSB tested that neither bound well to poly(dA) nor was able to stimulate DNA polymerase III holoenzyme elongation rates. Poly [d(A-T)] melting studies suggest that at least three of the plasmid encoded SSBs are better helix-destabilizing proteins than is the E. coli SSB protein.
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PMID:Single-stranded DNA binding proteins (SSBs) from prokaryotic transmissible plasmids. 200 32

The Mr = 38,300 polypeptide of the purified recombinant rat DNA polymerase beta served as an excellent substrate for protein kinase C (PKC) in vitro but not for the catalytic subunit of cAMP-dependent protein kinase. The phosphorylation by PKC resulted in inactivation of DNA polymerase beta activity, and recovery was achieved by dephosphorylation with alkaline phosphatase. Since the phosphorylated DNA polymerase beta was retained with use of a single-stranded DNA-cellulose column, inactivation might occur at a site different from that for the DNA binding. Amino acid sequence analysis of the phosphopeptides revealed that the phosphorylated sites were 2 serine residues at positions 44 and 55 from the NH2 terminus, either or both of which might be involved in the catalytic activity of DNA polymerase beta. Thus, the inactivation of the DNA repair enzyme, DNA polymerase beta, by PKC may be an important process in the modification of DNA metabolism in the nucleus through signal transduction processes.
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PMID:Inactivation of DNA polymerase beta by in vitro phosphorylation with protein kinase C. 204 Jun 2

L-Glutamic acid (gamma-4'-hydroxyanilide) (GHB) is oxidized by tyrosinase to a quinone which inhibits DNA polymerase, RNA polymerase, and mitochondrial energy production within mushrooms. It was previously shown that GHB can kill B16 melanoma cells in culture, but lacks cytotoxicity for nontyrosinase-containing cells. We have conjugated this drug to a superpotent melanotropic peptide and examined the bioactivity of this conjugate to melanoma cells. 4'-Hydroxyaniline was attached to glutamic acid at position 5 in the superpotent melanotropin fragment analogue, Ac-[Nle4, D-Phe7]alpha-MSH4-10-NH2. The melanotropin:anilide conjugate, Ac-[Nle4, Glu(gamma-4'-hydroxyanilide)5, D-Phe7]alpha-MSH4-10-NH2, was not cytotoxic to B16 or Cloudman S91 mouse melanoma cells in culture, as determined by cell counts and protein assays. Interestingly, we also found that GHB stimulated melanoma cell tyrosinase above control levels in both melanoma cell lines. In our study, GHB itself also was found not to be cytotoxic to B16 or S91 melanoma cells in culture. In the frog skin bioassay, the melanotropin conjugate was more potent than alpha-MSH or Ac-[Nle4, D-Phe7]alpha-MSH4-10 in stimulating melanosome dispersion. These results demonstrate that putative chemotherapeutic ligands can be incorporated into active-site fragment analogues of MSH without loss of biological activity.
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PMID:Synthesis and actions of a melanotropin conjugate, Ac-[Nle4, Glu(gamma-4'-hydroxyanilide)5, D-Phe7]alpha-MSH4-10-NH2, on melanocytes and melanoma cells in vitro. 216 79

Rat DNA polymerase beta (beta-pol) is a 39-kDa protein organized in two tightly folded domains, 8-kDa N-terminal and 31-kDa C-terminal domains, connected by a short protease-sensitive region. The 8-kDa domain contributes template binding to the intact protein, and we now report that the 31-kDa C-terminal domain contributes catalytic activity. Our results show that this domain as a purified proteolytic fragment conducts DNA synthesis under appropriate conditions but the kcat is lower and primer extension properties are different from those of the intact enzyme. A proteolytic truncation of the 31-kDa catalytic domain fragment, to remove a 60-residue segment from the NH2-terminal end, results in nearly complete loss of activity, suggesting the importance of this segment. Overall, these results indicate that the domains of beta-pol have distinct functional roles, template binding and nucleotidyltransferase, respectively; yet, the intact protein is more active for each function than the isolated individual domain fragment.
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PMID:Identification and properties of the catalytic domain of mammalian DNA polymerase beta. 220 97

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 functional domains of the avian retrovirus polymerase gene are at least tripartite in nature. Three enzymatic domains exist; the RNase H and DNA polymerase activities are located on the alpha subunit while the DNA endonuclease is located on the pp32 moiety. Virus mutants possessing deletions in the pp32 region demonstrated that this region encodes function(s) essential for replication of the virus while separate point mutations generated near the NH2 terminus of pp32 resulted in decreased replication and cell transformation. Molecular analysis of various steps in the virus replication cycle demonstrated that the synthesis of linear viral DNA, transport of viral DNA to the nucleus, and its subsequent circularization and integration into cellular DNA are apparently not affected in these point mutants. However, the synthesis of viral RNA from the integrated provirus of these point mutants appears less than that observed in wild type virus-infected cells. What role the mutated pp32 protein might have on viral transcription is discussed.
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PMID:Mutants of the Rous sarcoma virus reverse transcriptase gene are nondefective in early replication events. 240 84


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