Inhibition of adenovirus DNA polymerase by modified nucleoside triphosphate analogs correlate with their antiviral effects on cellular level

2000 ◽  
Vol 189 (2) ◽  
pp. 91-95 ◽  
Author(s):  
Renate Mentel ◽  
Stefan Kurek ◽  
Ursula Wegner ◽  
Martin v. Janta-Lipinski ◽  
Lutz Gürtler ◽  
...  
Keyword(s):  
Dna Polymerase ◽  
Cellular Level ◽  
Nucleoside Triphosphate ◽  
Antiviral Effects ◽  
Modified Nucleoside

scholarly journals The Structure of a High Fidelity DNA Polymerase Bound to a Mismatched Nucleotide Reveals an “Ajar” Intermediate Conformation in the Nucleotide Selection Mechanism

2011 ◽  
Vol 286 (22) ◽  
pp. 19758-19767 ◽  
Author(s):  
Eugene Y. Wu ◽  
Lorena S. Beese
Keyword(s):  
Dna Polymerase ◽  
Reaction Pathway ◽  
Insertion Site ◽  
Nucleoside Triphosphate ◽  
High Fidelity ◽  
Dna Polymerase I ◽  
Closed Conformation ◽  
Active Site Tyrosine ◽  
Pass Through ◽  
Polymerase I

To achieve accurate DNA synthesis, DNA polymerases must rapidly sample and discriminate against incorrect nucleotides. Here we report the crystal structure of a high fidelity DNA polymerase I bound to DNA primer-template caught in the act of binding a mismatched (dG:dTTP) nucleoside triphosphate. The polymerase adopts a conformation in between the previously established “open” and “closed” states. In this “ajar” conformation, the template base has moved into the insertion site but misaligns an incorrect nucleotide relative to the primer terminus. The displacement of a conserved active site tyrosine in the insertion site by the template base is accommodated by a distinctive kink in the polymerase O helix, resulting in a partially open ternary complex. We suggest that the ajar conformation allows the template to probe incoming nucleotides for complementarity before closure of the enzyme around the substrate. Based on solution fluorescence, kinetics, and crystallographic analyses of wild-type and mutant polymerases reported here, we present a three-state reaction pathway in which nucleotides either pass through this intermediate conformation to the closed conformation and catalysis or are misaligned within the intermediate, leading to destabilization of the closed conformation.

On the enzymatic incorporation of an imidazole nucleotide into DNA

2017 ◽  
Vol 15 (20) ◽  
pp. 4449-4455 ◽  
Author(s):  
Pascal Röthlisberger ◽  
Fabienne Levi-Acobas ◽  
Ivo Sarac ◽  
Philippe Marlière ◽  
Piet Herdewijn ◽  
...  
Keyword(s):  
Genetic Code ◽  
Base Pair ◽  
Nucleoside Triphosphate ◽  
Metal Base ◽  
Modified Nucleoside

We have evaluated the possibility for using an imidazole modified nucleoside triphosphate for the enzymatic construction of artificial metal base pair with view on an expansion of the genetic code.

scholarly journals Structural and Kinetic Analysis of Nucleoside Triphosphate Incorporation Opposite an Abasic Site by Human Translesion DNA Polymerase η

2015 ◽  
Vol 290 (13) ◽  
pp. 8028-8038 ◽  
Author(s):  
Amritaj Patra ◽  
Qianqian Zhang ◽  
Li Lei ◽  
Yan Su ◽  
Martin Egli ◽  
...  
Keyword(s):  
Kinetic Analysis ◽  
Dna Polymerase ◽  
Nucleoside Triphosphate ◽  
Abasic Site ◽  
Translesion Dna

Synergistic Antiviral Activity of Inhibitors of S-Adenosylhomocysteine Hydrolase and Ribavirin

1993 ◽  
Vol 4 (2) ◽  
pp. 127-130 ◽  
Author(s):  
H. Ishii ◽  
M. Hasobe ◽  
J. G. McKee ◽  
D. B. Ault-Riché ◽  
R. T. Borchardt
Keyword(s):  
Antiviral Activity ◽  
Synergistic Effects ◽  
Cellular Level ◽  
Carbocyclic Nucleosides ◽  
Viral Mrna ◽  
Antiviral Action ◽  
Cellular Toxicity ◽  
Imp Dehydrogenase ◽  
Adenosylhomocysteine Hydrolase ◽  
Antiviral Effects

(1′R,2′S,3′R)-9-(2′,3′-Dihydroxycycloperrt-4′-en-1′-yl)-adenine (DHCeA) and -3-deazaadenine (3-deaza-DHCeA), which are potent inhibitors of S-adenosylho-mocysteine (AdoHcy) hydrolase, and ribavirin, which is an inhibitor of IMP-dehydrogenase, were found in this study to have synergistic effects on inhibiting vaccinia virus replication in murine L929 cells without creating a synergistic effect on cellular toxicity. Thus, the antiviral effectiveness of this drug combination was 5–10× higher than the antiviral effectiveness observed with the AdoHcy hydrolase inhibitors alone. Ribavirin does not alter the ability of DHCeA and 3-deaza-DHCeA to elevate the intracellular AdoHcy/S-adeno-sylmethionine (AdoMet) ratio. Increases in this ratio were shown earlier to correlate with the antiviral effects of these carbocyclic nucleosides. Ribavirin was also shown to significantly reduce the cellular level of GTP, which is consistent with its activity as an inhibitor of IMP-dehydrogenase and its proposed mechanism of antiviral action, inhibiting the formation of the ‘capped methylated structure’ at the 5′-end of viral mRNA.

The mutant β E202K sliding clamp protein impairs DNA polymerase III replication activity

2021 ◽  
Author(s):  
Caleb Homiski ◽  
Michelle K. Scotland ◽  
Vignesh M. P. Babu ◽  
Sundari Chodavarapu ◽  
Robert W. Maul ◽  
...  
Keyword(s):  
Dna Replication ◽  
Dna Polymerase ◽  
Genetic Selection ◽  
Cellular Level ◽  
Dna Polymerase Iii ◽  
E Coli ◽  
Polymerase Iii ◽  
Pol Iii

Expression of the E. coli dnaN -encoded β clamp at ≥10-fold higher than chromosomally-expressed levels impedes growth by interfering with DNA replication. We hypothesized that the excess β clamp sequesters the replicative DNA polymerase III (Pol III) to inhibit replication. As a test of this hypothesis, we measured the ability of eight mutant clamps obtained by their inability to impede growth to stimulate Pol III replication in vitro . Compared with the wild type clamp, seven of the mutants were defective, consistent with their elevated cellular levels failing to sequester Pol III. However, the β E202K mutant, which bears a glutamic acid-to-lysine substitution at residue 202 displayed an increased affinity for Pol IIIα and Pol III core (Pol IIIαεθ), suggesting that it could still effectively sequester Pol III. Of interest, β E202K supported in vitro DNA replication by Pol II and Pol IV, but was defective with Pol III. Genetic experiments indicated that the dnaN E202K strain remained proficient in DNA damage-induced mutagenesis, but was modestly induced for SOS and displayed sensitivity to ultraviolet light and methyl methanesulfonate. These results correlate an impaired ability of the mutant β E202K clamp to support Pol III replication in vivo with its in vitro defect in DNA replication. Taken together, our results: (i) support the model that sequestration of Pol III contributes to growth inhibition, (ii) argue for existence of an additional mechanism that contributes to lethality and (iii) suggest that physical and functional interactions of the β clamp with Pol III are more extensive than currently appreciated. IMPORTANCE The β clamp plays critically important roles in managing the actions of multiple proteins at the replication fork. However, we lack a molecular understanding of both how the clamp interacts with these different partners, and the mechanisms by which it manages their respective actions. We previously exploited the finding that an elevated cellular level of the β clamp impedes E. coli growth by interfering with DNA replication. Using a genetic selection method, we obtained novel mutant β clamps that fail to inhibit growth. Their analysis revealed that β E202K is unique among them. Our work offers new insights into how the β clamp interacts with and manages the actions of E. coli DNA polymerases II, III and IV.

scholarly journals A C-Terminal Helicase Domain of the Human Papillomavirus E1 Protein Binds E2 and the DNA Polymerase α-Primase p68 Subunit

1998 ◽  
Vol 72 (9) ◽  
pp. 7407-7419 ◽  
Author(s):  
Philip J. Masterson ◽  
Margaret A. Stanley ◽  
Alan P. Lewis ◽  
Michael A. Romanos
Keyword(s):  
Human Papillomavirus ◽  
Dna Replication ◽  
Dna Polymerase ◽  
Deletion Analysis ◽  
Nucleoside Triphosphate ◽  
Structural Domain ◽  
Helicase Domain ◽  
Sequence Alignments ◽  
Hpv 16 ◽  
Dna Polymerase Α

ABSTRACT The human papillomavirus (HPV) E1 and E2 proteins bind cooperatively to the viral origin of replication (ori), forming an E1-E2-ori complex that is essential for initiation of DNA replication. All other replication proteins, including DNA polymerase α-primase (polα-primase), are derived from the host cell. We have carried out a detailed analysis of the interactions of HPV type 16 (HPV-16) E1 with E2, ori, and the four polα-primase subunits. Deletion analysis showed that a C-terminal region of E1 (amino acids [aa] 432 to 583 or 617) is required for E2 binding. HPV-16 E1 was unable to bind theori in the absence of E2, but the same C-terminal domain of E1 was sufficient to tether E1 to the ori via E2. Of the polα-primase subunits, only p68 bound E1, and binding was competitive with E2. The E1 region required (aa 397 to 583) was the same as that required for E2 binding but additionally contained 34 N-terminal residues. In confirmation of these differences, we found that a monoclonal antibody, mapping adjacent to the N-terminal junction of the p68-binding region, blocked E1-p68 but not E1-E2 binding. Sequence alignments and secondary-structure prediction for HPV-16 E1 and other superfamily 3 (SF3) viral helicases closely parallel the mapping data in suggesting that aa 439 to 623 constitute a discrete helicase domain. Assuming a common nucleoside triphosphate-binding fold, we have generated a structural model of this domain based on the X-ray structures of the hepatitis C virus and Bacillus stearothermophilus (SF2) helicases. The modelling closely matches the deletion analysis in suggesting that this region of E1 is indeed a structural domain, and our results suggest that it is multifunctional and critical to several stages of HPV DNA replication.

scholarly journals Study on Suitability of KOD DNA Polymerase for Enzymatic Production of Artificial Nucleic Acids Using Base/Sugar Modified Nucleoside Triphosphates

Molecules ◽  
2010 ◽  
Vol 15 (11) ◽  
pp. 8229-8240 ◽  
Author(s):  
Masayasu Kuwahara ◽  
Yuuki Takano ◽  
Yuuya Kasahara ◽  
Hiroki Nara ◽  
Hiroaki Ozaki ◽  
...  
Keyword(s):  
Nucleic Acids ◽  
Dna Polymerase ◽  
Enzymatic Production ◽  
Nucleoside Triphosphates ◽  
Modified Nucleoside

scholarly journals GS-9191 Is a Novel Topical Prodrug of the Nucleotide Analog 9-(2-Phosphonylmethoxyethyl)Guanine with Antiproliferative Activity and Possible Utility in the Treatment of Human Papillomavirus Lesions

2009 ◽  
Vol 53 (7) ◽  
pp. 2777-2784 ◽  
Author(s):  
Grushenka H. I. Wolfgang ◽  
Riri Shibata ◽  
Jianying Wang ◽  
Adrian S. Ray ◽  
Sylvia Wu ◽  
...  
Keyword(s):  
Human Papillomavirus ◽  
Dna Polymerase ◽  
Active Form ◽  
Nucleoside Triphosphate ◽  
Cottontail Rabbit ◽  
Nucleotide Analog ◽  
Infected Cells ◽  
Mitochondrial Dna Polymerase ◽  
Dna Polymerase Γ

ABSTRACT GS-9191 is a novel double prodrug of the nucleotide analog 9-(2-phosphonylmethoxyethyl)guanine (PMEG) designed as a topical agent to permeate skin and be metabolized to the active nucleoside triphosphate analog in the epithelial layer. The prodrug was shown to be metabolized intracellularly to 9-(2-phosphonylmethoxyethyl)-N6-cyclopropyl-2,6,diaminopurine (cPrPMEDAP) and subsequently deaminated to PMEG. The active form, PMEG diphosphate, was shown to be a potent inhibitor of DNA polymerase α and ß while showing weaker activity against mitochondrial DNA polymerase γ (50% enzyme inhibition observed at 2.5, 1.6, and 59.4 μM, respectively). GS-9191 was markedly more potent than PMEG or cPrPMEDAP in a series of human papillomavirus (HPV)-positive cell lines, with effective concentrations to inhibit 50% cell growth (EC50) as low as 0.03, 207, and 284 nM, respectively. In contrast, GS-9191 was generally less potent in non-HPV-infected cells and primary cells (EC50s between 1 and 15 nM). DNA synthesis was inhibited by GS-9191 within 24 h of treatment; cells were observed to be arrested in S phase by 48 h and to subsequently undergo apoptosis (between 3 and 7 days). In an animal model (cottontail rabbit papillomavirus), topical GS-9191 was shown to decrease the size of papillomas in a dose-related manner. At the highest dose (0.1%), cures were evident at the end of 5 weeks, and lesions did not recur in a 30-day follow-up period. These data suggest that GS-9191 may have utility in the treatment of HPV-induced lesions.

scholarly journals Yeast DNA polymerase   makes functional contacts with the DNA minor groove only at the incoming nucleoside triphosphate

2003 ◽  
Vol 100 (9) ◽  
pp. 5113-5118 ◽  
Author(s):  
M. T. Washington ◽  
W. T. Wolfle ◽  
T. E. Spratt ◽  
L. Prakash ◽  
S. Prakash
Keyword(s):  
Dna Polymerase ◽  
Minor Groove ◽  
Nucleoside Triphosphate ◽  
Dna Minor Groove
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