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:2.7.1.21 (thymidine kinase)
7,561 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Two non-conventional analogues of ATP, 3'-deoxyadenosine-2'-triphosphate (3'-d-2'-ATP) and 2'-deoxyadenosine-3'-triphosphate (2'-d-3'-ATP), the syntheses of which are described, were examined as potential phosphate donors for the nucleoside kinases: 2'-deoxycytidine kinase (dCK), cytosolic thymidine kinase (TK1) and mitochondrial thymidine kinase (TK2). The reactions were monitored by means of a mixture of [gamma-32P]ATP and cold analogue, and/or with the use of 3H-labelled acceptors and cold donor. With dCK, using equimolar mixtures of ATP with each analogue, and dC as acceptor, phosphate transfer from 3'-d-2'-ATP and 2'-d-3'-ATP amounted to 34% and 14%, respectively. With each analogue used alone (each at concentration of 100 microM), phosphate transfer from 3'-d-2'-ATP was 55% that from ATP, and from 2'-d-3'-ATP 16%. With human TK2, and equimolar mixtures of [gamma-32P]ATP with each of the analogues, and 1 microM dT as acceptor, there was no detectable transfer from either analogue. But, when each analogue was used alone, phosphate transfer attained 11% and 5%, respectively, that for ATP alone. With the low affinity form of human TK1, and dT as acceptor, only low phosphate transfer occurred with either analogue used alone. Both compounds exhibited Michaelis-Menten kinetics (with significantly lower Vmax than ATP), while ATP exhibited cooperative kinetics with all three kinases.
Acta Biochim Pol 1998
PMID:Unusual nucleoside triphosphate donors for nucleoside kinases: 3'-deoxyadenosine-2'-triphosphate and 2'-deoxyadenosine-3'-triphosphate. 970

DNA polymerases differentiate between correct and incorrect substrates during synthesis on undamaged DNA templates through the biochemical steps of base incorporation, primer-template extension and proofreading excision. Recent research examining DNA polymerase processing of abasic, alkylation and oxidative lesions is reviewed in light of these discrimination mechanisms. Inhibition of DNA synthesis results from correct polymerase discrimination against utilization of geometrically incorrect template bases or 3' terminal basepairs. The efficiency of translesion synthesis is thus related to the physical structure of the lesion containing DNA. However, variations in enzyme structure and kinetics result in translesion synthesis efficiencies that are also dependent upon the DNA polymerase. With a low probability, polymerase misinsertion events create a 3' lesion terminus which is geometrically favored over the correct lesion basepair, resulting in mutagenic translesion synthesis. For example, both polymerase alpha and polymerase beta appear to require the formation of a stable 3' primer-template structure for efficient abasic site translesion synthesis. However, the enzymes differ as to the precise molecular make-up of the stable DNA structure, resulting in different mutational specificities. Similar mechanisms may be applicable to oxidative damage, where mutational specificities dependent upon the DNA polymerase also have been observed. In vitro reaction conditions also influence DNA polymerase processing of lesions. Using an in vitro herpes simplex virus thymidine kinase (HSV-tk) gene forward mutation assay, we demonstrate that high dNTP substrate concentrations affect the mutagenic specificity of translesion synthesis using alkylated templates. The exonuclease-deficient Klenow polymerase error frequency for G-->A transition mutations using templates modified by N-ethyl-N-nitrosourea (ENU) was four-fold higher at 1000 microM [dNTP], relative to 50 microM [dNTP], consistent with an increased efficiency of extension of the etO6G.T mispair. Moreover, the frequency of other ENU-induced polymerase errors was suppressed when polymerase reactions contained 50 microM dNTP, relative to 1000 microM dNTP. The efficiency of proofreading as a polymerase error discrimination mechanism reflects a balance between the competing processes of 3'-->5' exonuclease removal of mispairs and polymerization of the next correct nucleotide. Polymerases that are devoid of a proofreading exonuclease generally display enhanced abasic site translesion synthesis relative to proofreading-proficient enzymes. In addition, the proofreading exonucleases of Escherichia coli Pol I and T4 DNA polymerases have been found to remove mispairs caused by abasic sites and oxidative lesions, respectively, resulting in lowered polymerase error rates. However, the magnitude of the exonuclease effect is small (less than 10-fold), and highly dependent upon the DNA polymerase-exonuclease. We have studied proofreading exonuclease removal of alkylation damage in the HSV-tk forward assay. We observed no significant reduction in the magnitude of the mutant frequency vs. dose-response curves when N-methyl-N-nitrosourea or ENU-treated templates were used in exonuclease-proficient Klenow polymerase reactions, as compared to the exonuclease-deficient polymerase reactions. Thus, available data suggest that proofreading excision of endogenous lesion mispairs does occur, but the efficiency is dependent upon the lesion and the DNA polymerase-exonuclease studied.
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PMID:DNA polymerase mutagenic bypass and proofreading of endogenous DNA lesions. 1006 63

Copolymers of N-polyvinylpyrrolidone-acrylic acid (AB-1) and adamantane derivatives are known to possess marked antiviral activity in in vitro and in ovo models. Among the constructed preparations of AB-1 modified by adamantane derivatives some, especially AB-4 (modified by deitiforin), were found to show more extended antiviral activity and to inhibit markedly virus reproduction in susceptible permissive cell cultures and chicken embryos. In AB-4 treated cells and allantoic sacs, virus titers (influenza virus, herpes virus, and HIV) and virus antigen concentration were decreased. On the other hand, herpes virus-specific thymidine kinase and of DNA-polymerases isolated from Escherichia coli, Plectonema boryanum, and herpes virus type 1 infected murine brain tissue retained their activity after incubation with AB-4 or AB-2. The compounds investigated, in view of their effect on virus reproduction, are thought to be prospective as antiviral agents.
Acta Biochim Pol 2001
PMID:Therapeutical effect of modified adamantane copolymer compounds: study of molecular mechanisms. 1144 Jan 76

In vitro susceptibility assays of herpes simplex virus (HSV) do not necessarily correlate with treatment outcome. An HSV type 1 (HSV-1) isolate, N4, recovered from a patient who presented with herpes keratitis with localized immunosuppression, was characterized for susceptibility. Although the 50% inhibitory concentration (IC(50)) for this isolate was less than the accepted breakpoint for defining resistance to acyclovir (>2.0 microg/mL), the following lines of evidence suggest that the isolate was acyclovir resistant: (1) the clinical history confirmed that the infection was nonresponsive to acyclovir; (2) the in vitro susceptibility was similar to that of a thymidine kinase (TK)-negative, acyclovir-resistant virus SLU360; (3) the IC(50) of acyclovir was more than 10 times the IC(50) for an acyclovir-susceptible control strain; (4) plaque-purified clonal isolates were resistant to acyclovir (IC(50)s, >2.0 microg/mL); and (5) biochemical studies indicated that the HSV-1 N4 TK was partially impaired for acyclovir phosphorylation. Although residue changes were found in both the viral tk and pol coding regions of HSV-1 N4, characterization of a recombinant virus expressing the HSV-1 N4 polymerase suggested that the TK and Pol together conferred the acyclovir-resistance phenotype.
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PMID:Biochemical characterization of a virus isolate, recovered from a patient with herpes keratitis, that was clinically resistant to acyclovir. 1171 95

A total of 21 clones of acyclovir (ACV)-resistant (ACV(r)) herpes simplex virus type 1 (HSV-1) and 23 clones of penciclovir (PCV)-resistant (PCV(r)) HSV-1, emerging during serial passages in the presence of ACV or PCV, were isolated under conditions excluding contamination of resistant mutants in the starting virus culture, and their mutations in the thymidine kinase (TK) and DNA polymerase (DNA Pol) genes were analyzed comparatively. Mutations in the TK genes from ACV(r) mutants consisted of 50% single nucleotide substitutions and 50% frameshift mutations, while the corresponding figures for the PCV(r) mutants were 4 and 96%, respectively (P < 0.001). Eight of the 21 ACV(r) clones, but none of the 23 PCV(r) clones, had mutations in DNA Pol. Only nucleotide substitution(s) could be detected in the DNA Pol gene, as the gene is essential for virus replication. Therefore, the results for the DNA Pol mutants are concordant with those for the TK mutants in that a single nucleotide substitution was commonly observed in the ACV(r), but not in the PCV(r), mutants. These results clearly point to differential mutation patterns between ACV(r) and PCV(r) HSV-1 clones.
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PMID:Differential mutation patterns in thymidine kinase and DNA polymerase genes of herpes simplex virus type 1 clones passaged in the presence of acyclovir or penciclovir. 1270 44

We have examined the kinetics of incorporation of acyclovir triphosphate by the herpes simplex virus-1 DNA polymerase holoenzyme (Pol-UL42) and the human mitochondrial DNA polymerase using transient kinetic methods. For each enzyme, we compared the kinetic parameters for acyclovir to those governing incorporation of dGTP. The favorable ground state dissociation constant (6 microM) and rate of polymerization (10 s(-1)) afford efficient incorporation of acyclovir triphosphate by the Pol-UL42 enzyme. A discrimination factor of approximately 50 favors dGTP over acyclovir triphosphate, mostly due to a faster maximum rate of dGTP incorporation. Once incorporated, acyclovir is removed with a half-life of approximately 1 h in the presence of a normal concentration of deoxynucleoside triphosphates, leading to a high toxicity index (16,000) toward viral replication. To assess the potential for toxicity toward the host we examined the incorporation and removal of acyclovir triphosphate by the human mitochondrial DNA polymerase. These results suggest moderate inhibition of mitochondrial DNA replication defining a toxicity index of 380. This value is much higher than the value of 1.5 determined for tenofovir, another acyclic nucleoside analog. The enzymatic therapeutic index is only 42 in favoring inhibition of the viral polymerase over polymerase gamma, whereas that for tenofovir is greater than 1,200. Mitochondrial toxicity is relatively low because acyclovir is activated only in infected cells by the promiscuous viral thymidine kinase and otherwise, mitochondrial toxicity would accumulate during long term treatment.
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PMID:Enzymatic therapeutic index of acyclovir. Viral versus human polymerase gamma specificity. 1757 51

ICP4 is the major activator of herpes simplex virus (HSV) transcription. Previous studies have defined several regions of ICP4 that are important for viral gene expression, including a DNA binding domain and transactivation domains that are contained in the C-terminal and N-terminal 520 and 274 amino acids, respectively. Here we show that the N-terminal 210 amino acids of ICP4 are required for interactions with components of TFIID and mediator and, as a consequence, are necessary for the activation of viral genes. A mutant of ICP4 deleted for amino acids 30 to 210, d3-10, was unable to complement an ICP4 null virus at the level of viral replication. This was the result of a severe deficiency in viral gene and protein expression. The absence of viral gene expression coincided with a defect in the recruitment of RNA polymerase II to a representative early promoter (thymidine kinase [TK]). Affinity purification experiments demonstrated that d3-10 ICP4 was not found in complexes with components of TFIID and mediator, suggesting that the defect in RNA polymerase II (Pol II) recruitment was the result of ablated interactions between d3-10 and TFIID and mediator. Complementation assays suggested that the N-terminal and C-terminal regions of ICP4 cooperate to mediate gene expression. The complementation was the result of the formation of more functional heterodimers, which restored the ability of the d3-10-containing molecules to interact with TFIID. Together, these studies suggest that the N terminus contains a true activation domain, mediating interactions with TFIID, mediator, and perhaps other transcription factors, and that the C terminus of the molecule contains activities that augment the functions of the activation domain.
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PMID:Requirement of the N-terminal activation domain of herpes simplex virus ICP4 for viral gene expression. 2313 15

Herpes simplex virus (HSV) resistance to antivirals constitutes a therapeutic challenge, especially among immunocompromised patients. This observational survey on HSV resistance to antivirals was conducted retrospectively over a 4-year period (2008-2012). A total of 211 HSV-positive clinical samples (94 HSV-1 and 117 HSV-2) recovered from 139 patients (11 immunocompetent patients, 85 immunocompromised patients, and 43 patients with unknown immune status) with suspected HSV drug-resistance were analyzed for acyclovir and foscarnet susceptibility. Antiviral resistance testing consisted in a two-step procedure including a first-step genotypic assay, based on UL23 (thymidine kinase, TK) and UL30 (Pol) gene sequencing, and a second-step phenotypic assay (i.e., plaque reduction assay) performed when unpreviously described mutations were detected. As a whole, susceptibility and resistance to antivirals were evidenced for 58 (30.7%) and 86 (45.5%) HSV, respectively, whereas antiviral profile remained undetermined for 45 (23.8%) HSV. The prevalence of drug resistance was significantly higher among HSV-2 isolates than among HSV-1 isolates (53.8% vs. 34.9%; p=0.012). The majority (i.e., 79.7%) of cases of ACV resistance conferred by TK mutations resulted from UL23 gene frameshift reading. Apart from the changes surely related to natural polymorphism or drug-resistance, 91 unpreviously reported mutations were identified in TK and Pol, including 51 potential natural polymorphisms, 22 mutations likely conferring resistance to antivirals, and 18 mutations of unclear significance.
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PMID:Surveillance of herpes simplex virus resistance to antivirals: a 4-year survey. 2407 63

Adjuvant chemotherapy with 5-fluorouracil remains the basic treatment for patients with advanced colorectal carcinoma. The major obstacle in successful treatment is the ability of CRC cells to acquire chemoresistance. Here we examined the impact of ID1 silencing on the sensitivity of CRC cells to 5-FU. To suppress ID1 expression in HT-29 and HCT-116 cells the cells were transduced with a lentiviral vector carrying the ID1 silencing sequence. Cells with silenced ID1 showed altered expression of epithelial and mesenchymal markers and exhibited increased proliferation rate compared to the parental cells. HCT-116 cells with suppressed ID1 became sensitized to 5-FU and this was not observed in HT-29 cells. Silencing ID1 resulted in altered expression of genes encoding enzymes metabolizing 5-FU. HT-29 cells with suppressed ID1 had significantly reduced mRNA level for thymidine phosphorylase, uridine-cytydine kinase 2 and dihydropyrimidine dehydrogenase. ID1 suppression in HCT-116 cells resulted in an increase of mRNA level for thymidine phosphorylase, thymidine kinase and uridine-cytydine kinase 2 with concurrent drop of dihydropyrimidine dehydrogenase and thymidylate synthetase mRNA levels. In conclusion, ID1 expression impacts the sensitivity of colon cancer cells to 5-FU and may be considered as a potential predictive marker in CRC treatment.
Acta Biochim Pol 2017
PMID:Suppression of ID1 expression in colon cancer cells increases sensitivity to 5-fluorouracil. 2851 Jun 12


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