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

The modification of Klenow fragment of DNA polymerase I E. coli was investigated by the affinity reagents d(Tp)2C[Pt2+(NH3)2OH](pT)7 and d(pT)2pC[Pt2+(NH3)2OH](pT)7. The template binding site of the enzyme was modified by these reagents in the presence of NaF (5 mM), which inhibits selectively the 3'----5'-exonuclease activity of the enzyme and therefore prevents the reagent from degradation. NaCN destroyed covalent bonds between reagents and enzyme, restoring activity of the Klenow fragment. The affinity of different ligands (inorganic phosphate, nucleoside monophosphates, oligonucleotides) to the template binding site of Klenow fragment was estimated. Minimal ligands capable to bind with the template site were shown to be triethylphosphate (Kd 290 microM) and phosphate (Kd 26 microM). Ligand affinity increases by the factor 1.76 per an added (monomer unit from phosphate to d(pT) and then for oligonucleotides d(Tp)nT (n 1 to 19-20). At n greater than 19-20, the ligand affinity remained constant. The complete ethylation of phosphodiester groups lowers affinity of the oligothymidylates to the enzyme by approximately 10 times, and comparable decrease of Pt2+-oligonucleotide affinity to polymerase is caused by the absence of Mn2+-ions. The data obtained led to suggestion that one Me2+-dependent electrostatic contact of the template phosphodiester group with the enzyme takes place (delta G = -1.45...-1.75 kcal/mole). Formation of a hydrogen bond with the oxygen atom of P = O group of the same template phosphate is also assumed (delta G = -4.8...-4.9 kcal/mole). Other template internucleotide phosphates do not interact with the enzyme but the bases of oligonucleotides take part in hydrophobic interactions with the template binding site. Gibbs energy changes by -0.34 kcal/mole when the template is lengthened by one unit.
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PMID:[Klenow fragment of DNA-polymerase I from E. coli. III. The role of internucleotide phosphate groups of the matrix in its binding with the enzyme]. 266 77

The values of Kd and Gibbs energy (delta G degrees) have been measured for complexes of the template site of DNA polymerase I Klenow fragment with the homo-oligonucleotides d(pC)n, d(pT)n, and d(pA)n and hetero-oligonucleotides of various structures and lengths. These parameters were evaluated from the protective effect of the oligonucleotide on enzyme inactivation by the affinity reagents d(Tp)2C[Pt2+ (NH3)2OH](pT)7 and d[(Tp2)C(Pt2+(NH3)2OH)p]3T of the template site. The present results and previously reported data [(1985) Biorg. Khim. 13, 357-369] indicate that the nucleoside components of the template form complexes as a result of their hydrophobic interactions with the enzyme. Only one template internucleotide phosphate forms an Me2+-dependent electrostatic contact and a hydrogen bond with the enzyme. The 19-20-nucleotide fragments of the template appear to interact with the protein molecule.
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PMID:DNA polymerase I (Klenow fragment): role of the structure and length of a template in enzyme recognition. 272 84

The affinity of different ligands (phosphate, nucleoside monophosphates, oligonucleotides) to the template binding site of DNA polymerase alpha from human placenta was estimated. To this goal, dependences of rate of the enzyme inactivation by the affinity reagent d(pT)2pC[Pt2+(NH3)2OH](pT)7 on the concentration of these ligands as competitive inhibitors were determined. Minimal ligands capable to bind with the template site of DNA polymerase alpha were shown to be triethylphosphate (Kd 600 microM) and phosphate (Kd 53 microM). Ligand affinity increases by the factor 1.71 per added monomer unit from phosphate to d(pT) and then for oligothymidylates d(Tp)nT (n 1 to 14). The partial ethylation of phosphodiester groups does not change the efficiency of the oligothymidylate binding with the enzyme. However, the complete ethylation of these groups lowers affinity of the oligothymidylates to the enzyme by 7-9 times. The decrease is comparable with the change of Pt2+-decathymidylate affinity to the enzyme caused by Mn2+-ions. The data obtained led to suggestion that an electrostatic contact (most likely, Me2+-dependent) of phosphodiester group with the enzyme takes place. The type of contact is confirmed by Gibbs' energy change 1.1-1.4 kcal/mole. Formation of a hydrogen bond with the oxygen atom of P = O group of the same phosphate is also assumed (delta G =--4.4 . . .--4.5 kcal/mole). The other internucleotide phosphates and all bases of oligonucleotides form neither hydrogen bonds nor electrostatic contacts with the template binding site. Gibbs' energy changes by 0.32 kcal/mole when the template is lengthened by one unit. We suppose that this value characterizes the energy gain in the transition of oligonucleotide template from aquous medium to the hydrophobic environement of the enzyme active site. Comparison of Km values of oligothymidylates and their partially or completely ethylated analogues as templates in the reaction of DNA polymerization catalysed by DNA polymerase alpha from human placenta and Klenow's fragment of E. coli DNA polymerase I suggests a similar mechanism of template recognition by both enzymes.
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PMID:[Eukaryotic and prokaryotic DNA-polymerase. II. The role of internucleotide phosphate groups of a template in its binding with the enzyme]. 355 64

Affinity labelling of human placenta DNA polymerase alpha (EC 2.7.7.7) with the reactive oligodeoxyribonucleotide d(pT)2pC[Pt2+(NH3)2OH](pT)7 was used for quantitative analysis of enzyme interaction with oligodeoxyribonucleotides as templates. Dissociation constants and Gibb's energy values for different oligothymidylates d(pT)nT where n = 1-14 have been evaluated by competitive experiments of these ligands with Pt2+ reagent. The data obtained prove the formation of one Me2+-dependent electrostatic contact and a hydrogen bond between the enzyme and one phosphate of these templates. One may suppose that the hydrophobic interaction of any other monomeric link of oligodeoxyribonucleotides with the enzyme template site takes place.
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PMID:Role of nucleoside components and internucleotide phosphate groups of oligodeoxyribonucleotide template in its binding to human DNA polymerase alpha. 358 74

Modification of human placenta DNA polymerase alpha by (pT)2pC[Pt2 + (NH3)2OH].(pT)7 was investigated. The linear time dependence of the enzyme activity logarithm suggested a pseudo-first order for modification. Kd value of enzyme-affinity reagent complex (0.5 microM) was estimated. The enzyme inactivation by the affinity reagent and protection from inactivation in the presence of oligonucleotides of varying length were used for determining Kd values of the enzyme-ligand complexes. Oligonucleotide d(pT)2pC(pT)7 (Kd 0.15 microM), d(Tp)9T (Kd 0.15 microM) and [d(Tp)9]ddT (Kd 0.15 microM) protected the enzyme from inactivation with equal efficiency. The protective action of oligothymidylates d(Tp)nT (where n changes from 3 to 14) strongly depended on the chain length, the Kd values diminishing from 5.3 to 0.0091 microM in the geometrical progression. The addition of one link to the oligothymidylate chain resulted in 1.71-fold increase in the oligonucleotide affinity for the enzyme specific site. Such a change corresponds to Gibbs energy change of about 0.32 kcal/mole. It is supposed that the monomer units of pentadecathymidylate (at least beginning with the third one) in d(Tp)14T-enzyme complex form neither hydrogen bonds nor electrostatic linkages with the enzyme. Kd values of oligonucleotides as templates are shown to reflect quite well the true affinity of template for the enzyme. This affinity increases in the presence of a primer. However, the ratio of the affinity for different oligonucleotides does not change in the presence or absence of a complementary primer.
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PMID:[DNA-polymerase alpha from human placenta. Effectiveness of interaction between oligothymidylates of different lengths and the template-binding site]. 396 8

N2-(p-n-Butylphenyl)-2'-deoxyguanosine (BuPdG) and its 5'-triphosphate (BuPdGTP), expected to be inhibitors of eukaryotic DNA polymerase alpha, have been synthesized. BuPdG was synthesized by two methods and characterized by 1H NMR and by chemical relation to guanosine. Direct synthesis involving silylated N2-(p-n-butylphenyl)guanine (BuPG) and 1-chloro-3,5-di-p-toluoyl-2-deoxyribofuranose in the presence of trimethylsilyl trifluoromethanesulfonate gave one alpha and two beta isomers of deoxyribonucleoside as determined by 1H NMR. However, NMR and UV spectra were equivocal in distinguishing between 7 and 9 isomers. The identity of the desired 9-beta-BuPdG was ultimately proved by its independent synthesis from the corresponding ribonucleoside. 1H NMR spectra of the O'-acetylated ribonucleosides of BuPG showed characteristic patterns of O'-acetylated guanosines, and their identity was proved by relating the products of the reaction of isomeric O'-acetylated 2-bromoinosines with p-n-butylaniline and with ammonia: the 2-bromoinosine which gave guanosine also gave the suspected 9-beta-ribonucleoside, BuPGr, and that which gave N7-beta-ribofuranosylguanine also gave the 7-beta isomer of BuPGr. BuPGr was transformed in a multistep procedure to give BuPdG, identical with the major beta isomer obtained by direct deoxynucleoside synthesis. The 5'-monophosphate of BuPdG was obtained by treatment of the nucleoside with phosphoryl chloride in trimethyl phosphate; the monophosphate reacted as the phosphoimidazolyl derivative with pyrophosphate to yield the 5'-triphosphate, BuPdGTP.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Synthesis and characterization of N2-(p-n-butylphenyl)-2'-deoxyguanosine and its 5'-triphosphate and their inhibition of HeLa DNA polymerase alpha. 669 66

The reaction between trans-diamminedichloroplatinum(II) and single-stranded oligonucleotides containing the sequence d(GXG) (X being an adenine, cytosine or thymine residue) yields trans-[Pt(NH3)2[(GXG)-GN7,GN7]] intrastrand cross-links. These cross-links do not prevent the pairing of the platinated oligonucleotides with their complementary strands but they decrease the thermal stability of the duplexes. The thermal stability is not much affected by the chemical nature of the X residue and its complementary base. By gel electrophoresis, it is shown that the trans- [Pt(NH3)2[d(GTG)-GN7,GN7]] cross-link bends the DNA double helix (26 degrees) and unwinds it (45 degrees). The pairing of the platinated oligonucleotides with their complementary strands promotes the rearrangement of the 1,3-intrastrand cross-links into interstrand cross-links. At a given temperature, the nature of the X residue, its complementary base and of the base pairs adjacent to the adducts do not dramatically affect the rate of the reaction. To know whether trans-[Pt(NH3)2[d(GXG)-GN7,GN7]] cross-links do not rearrange in some sequences, the location of these adducts was searched in double-stranded DNA after reaction with trans-diamminedichloroplatinum(II) by means of the 3'-5' exonuclease activity of T4 DNA polymerase. At low level of platination, trans-[Pt(NH3)2[d(GXG)-GN7,GN7]] cross-links were not detected. Monofunctional adducts and interstrand cross-links were mainly formed. These results are discussed in relation with the clinical inefficiency of trans-diamminedichloroplatinum(II).
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PMID:Intrastrand cross-links are not formed in the reaction between transplatin and native DNA: relation with the clinical inefficiency of transplatin. 763 Jul 15


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