scholarly journals Effect of incorporation of cidofovir into DNA by human cytomegalovirus DNA polymerase on DNA elongation.

1997 ◽  
Vol 41 (3) ◽  
pp. 594-599 ◽  
Author(s):  
X Xiong ◽  
J L Smith ◽  
M S Chen
Keyword(s):  
Dna Synthesis ◽  
Dna Polymerase ◽  
Human Cytomegalovirus ◽  
Chain Elongation ◽  
Exonuclease Activity ◽  
Dna Chain ◽  
Alternate Substrate

Cidofovir (CDV) (HPMPC) has potent in vitro and in vivo activity against human cytomegalovirus (HCMV), CDV diphosphate (CDVpp), the putative antiviral metabolite of CDV, is an inhibitor and an alternate substrate of HCMV DNA polymerase. CDV is incorporated with the correct complementation to dGMP in the template, and the incorporated CDV at the primer end is not excised by the 3'-to-5' exonuclease activity of HCMV DNA polymerase. The incorporation of a CDV molecule causes a decrease in the rate of DNA elongation for the addition of the second natural nucleotide from the singly incorporated CDV molecule. The reduction in the rate of DNA (36-mer) synthesis from an 18-mer by one incorporated CDV is 31% that of the control. However, the fidelity of HCMV DNA polymerase is maintained for the addition of the nucleotides following a single incorporated CDV molecule. The rate of DNA synthesis by HCMV DNA polymerase is drastically decreased after the incorporation of two consecutive CDV molecules; the incorporation of a third consecutive CDV molecule is not detectable. Incorporation of two CDV molecules separated by either one or two deoxynucleoside monophosphates (dAMP, dGMP, or dTMP) also drastically decreases the rate of DNA chain elongation by HCMV DNA polymerase. The rate of DNA synthesis decreases by 90% when a template which contains one internally incorporated CDV molecule is used. The inhibition by CDVpp of DNA synthesis by HCMV DNA polymerase and the inability of HCMV DNA polymerase to excise incorporated CDV from DNA may account for the potent and long-lasting anti-CMV activity of CDV.

DNA polymerase I proofreading exonuclease activity is required for endonuclease V repair pathway both in vitro and in vivo

DNA Repair ◽  
2018 ◽  
Vol 64 ◽  
pp. 59-67 ◽  
Author(s):  
Kang-Yi Su ◽  
Liang-In Lin ◽  
Steven D. Goodman ◽  
Rong-Syuan Yen ◽  
Cho-Yuan Wu ◽  
...  
Keyword(s):  
Dna Polymerase ◽  
Exonuclease Activity ◽  
Repair Pathway ◽  
Dna Polymerase I ◽  
Endonuclease V ◽  
Polymerase I

scholarly journals Dysfunctional proofreading in the Escherichia coli DNA polymerase III core

2004 ◽  
Vol 384 (2) ◽  
pp. 337-348 ◽  
Author(s):  
Duane A. LEHTINEN ◽  
Fred W. PERRINO
Keyword(s):  
Dna Polymerase ◽  
Gel Filtration ◽  
Exonuclease Activity ◽  
Dna Polymerase Iii ◽  
Wild Type ◽  
Α Subunit ◽  
Polymerase Iii ◽  
Pol Iii

The ε-subunit contains the catalytic site for the 3′→5′ proofreading exonuclease that functions in the DNA pol III (DNA polymerase III) core to edit nucleotides misinserted by the α-subunit DNA pol. A novel mutagenesis strategy was used to identify 23 dnaQ alleles that exhibit a mutator phenotype in vivo. Fourteen of the ε mutants were purified, and these proteins exhibited 3′→5′ exonuclease activities that ranged from 32% to 155% of the activity exhibited by the wild-type ε protein, in contrast with the 2% activity exhibited by purified MutD5 protein. DNA pol III core enzymes constituted with 11 of the 14 ε mutants exhibited an increased error rate during in vitro DNA synthesis using a forward mutation assay. Interactions of the purified ε mutants with the α- and θ-subunits were examined by gel filtration chromatography and exonuclease stimulation assays, and by measuring polymerase/exonuclease ratios to identify the catalytically active ε511 (I170T/V215A) mutant with dysfunctional proofreading in the DNA pol III core. The ε511 mutant associated tightly with the α-subunit, but the exonuclease activity of ε511 was not stimulated in the α–ε511 complex. Addition of the θ-subunit to generate the α–ε511–θ DNA pol III core partially restored stimulation of the ε511 exonuclease, indicating a role for the θ-subunit in co-ordinating the α–ε polymerase–exonuclease interaction. The α–ε511–θ DNA pol III core exhibited a 3.5-fold higher polymerase/exonuclease ratio relative to the wild-type DNA pol III core, further indicating dysfunctional proofreading in the α–ε511–θ complex. Thus the ε511 mutant has wild-type 3′→5′ exonuclease activity and associates physically with the α- and θ-subunits to generate a proofreading-defective DNA pol III enzyme.

scholarly journals Deoxyribonucleic acid synthesis in mammalian nuclei. Incorporation of deoxyribonucleotides and chain-terminating nucleotide analogues

1971 ◽  
Vol 121 (5) ◽  
pp. 803-809 ◽  
Author(s):  
M. A. Waqar ◽  
L. A. Burgoyne ◽  
M. R. Atkinson
Keyword(s):  
Dna Synthesis ◽  
Deoxyribonucleic Acid ◽  
Acid Synthesis ◽  
Deoxyribonucleic Acid Synthesis ◽  
Dna Chain ◽  
Relationship Of ◽  
The Relationship ◽  
Neighbour Analysis

The properties of a nuclear preparation from rat liver and thymus are described. (1) Nearest-neighbour analysis after incorporation of 32P-labelled nucleotide residues from dATP, dCTP, dGTP, dTTP and arabinofuranosyl analogues of CTP and ATP shows template-dependent DNA synthesis. (2) Where primer termini are limiting, incorporation of arabinofuranosyl analogues of AMP and CMP residues proceeds to a limit indicating that both of these analogues are DNA chain terminators. (3) No large differences have been found between the priming potentialities or the intrinsic DNA polymerase activities of nuclei from resting or regenerating liver and the relationship of this DNA synthesis in vitro to DNA replication or repair in vivo is briefly discussed.

scholarly journals Lobucavir is phosphorylated in human cytomegalovirus-infected and -uninfected cells and inhibits the viral DNA polymerase.

1997 ◽  
Vol 41 (12) ◽  
pp. 2680-2685 ◽  
Author(s):  
D J Tenney ◽  
G Yamanaka ◽  
S M Voss ◽  
C W Cianci ◽  
A V Tuomari ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Dna Synthesis ◽  
Dna Polymerase ◽  
Human Cytomegalovirus ◽  
Potent Inhibitor ◽  
Viral Dna ◽  
Infected Cells ◽  
Simplex Virus ◽  
Hsv Thymidine Kinase

Lobucavir (LBV) is a deoxyguanine nucleoside analog with broad-spectrum antiviral activity. LBV was previously shown to inhibit herpes simplex virus (HSV) DNA polymerase after phosphorylation by the HSV thymidine kinase. Here we determined the mechanism of action of LBV against human cytomegalovirus (HCMV). LBV inhibited HCMV DNA synthesis to a degree comparable to that of ganciclovir (GCV), a drug known to target the viral DNA polymerase. The expression of late proteins and RNA, dependent on viral DNA synthesis, was also inhibited by LBV. Immediate-early and early HCMV gene expression was unaffected, suggesting that LBV acts temporally coincident with HCMV DNA synthesis and not through cytotoxicity. In vitro, the triphosphate of LBV was a potent inhibitor of HCMV DNA polymerase with a Ki of 5 nM. LBV was phosphorylated to its triphosphate form intracellularly in both infected and uninfected cells, with phosphorylated metabolite levels two- to threefold higher in infected cells. GCV-resistant HCMV isolates, with deficient GCV phosphorylation due to mutations in the UL97 protein kinase, remained sensitive to LBV. Overall, these results suggest that LBV-triphosphate halts HCMV DNA replication by inhibiting the viral DNA polymerase and that LBV phosphorylation can occur in the absence of viral factors including the UL97 protein kinase. Furthermore, LBV may be effective in the treatment of GCV-resistant HCMV.

scholarly journals Palm Mutants in DNA Polymerases α and η Alter DNA Replication Fidelity and Translesion Activity

2004 ◽  
Vol 24 (7) ◽  
pp. 2734-2746 ◽  
Author(s):  
Atsuko Niimi ◽  
Siripan Limsirichaikul ◽  
Shonen Yoshida ◽  
Shigenori Iwai ◽  
Chikahide Masutani ◽  
...  
Keyword(s):  
Dna Synthesis ◽  
Dna Polymerase ◽  
Dna Polymerases ◽  
Pyrimidine Dimer ◽  
Replication Fidelity ◽  
Dna Polymerase Α ◽  
Replication Errors ◽  
Translesion Dna

ABSTRACT We isolated active mutants in Saccharomyces cerevisiae DNA polymerase α that were associated with a defect in error discrimination. Among them, L868F DNA polymerase α has a spontaneous error frequency of 3 in 100 nucleotides and 570-fold lower replication fidelity than wild-type (WT) polymerase α. In vivo, mutant DNA polymerases confer a mutator phenotype and are synergistic with msh2 or msh6, suggesting that DNA polymerase α-dependent replication errors are recognized and repaired by mismatch repair. In vitro, L868F DNA polymerase α catalyzes efficient bypass of a cis-syn cyclobutane pyrimidine dimer, extending the 3′ T 26,000-fold more efficiently than the WT. Phe34 is equivalent to residue Leu868 in translesion DNA polymerase η, and the F34L mutant of S. cerevisiae DNA polymerase η has reduced translesion DNA synthesis activity in vitro. These data suggest that high-fidelity DNA synthesis by DNA polymerase α is required for genomic stability in yeast. The data also suggest that the phenylalanine and leucine residues in translesion and replicative DNA polymerases, respectively, might have played a role in the functional evolution of these enzyme classes.

DNA Polymerase Fidelity: From Genetics Toward a Biochemical Understanding

Genetics ◽  
1998 ◽  
Vol 148 (4) ◽  
pp. 1475-1482
Author(s):  
Myron F Goodman ◽  
D Kuchnir Fygenson
Keyword(s):  
Dna Synthesis ◽  
Dna Polymerase ◽  
Bacteriophage T4 ◽  
Theoretical Models ◽  
Model Systems ◽  
Polymerase Fidelity ◽  
Base Selection ◽  
Physical Biochemistry

Abstract This review summarizes mutagenesis studies, emphasizing the use of bacteriophage T4 mutator and antimutator strains. Early genetic studies on T4 identified mutator and antimutator variants of DNA polymerase that, in turn, stimulated the development of model systems for the study of DNA polymerase fidelity in vitro. Later enzymatic studies using purified T4 mutator and antimutator polymerases were essential in elucidating mechanisms of base selection and exonuclease proofreading. In both cases, the base analogue 2-aminopurine (2AP) proved tremendously useful—first as a mutagen in vivo and then as a probe of DNA polymerase fidelity in vitro. Investigations into mechanisms of DNA polymerase fidelity inspired theoretical models that, in turn, called for kinetic and thermodynamic analyses. Thus, the field of DNA synthesis fidelity has grown from many directions: genetics, enzymology, kinetics, physical biochemistry, and thermodynamics, and today the interplay continues. The relative contributions of hydrogen bonding and base stacking to the accuracy of DNA synthesis are beginning to be deciphered. For the future, the main challenges lie in understanding the origins of mutational hot and cold spots.

Tokishakuyakusan Effect on DNA Polymerase α Activity in Relationship to DNA Synthesis before and/or after the LH/FSH Surge in Rats

1995 ◽  
Vol 23 (03n04) ◽  
pp. 231-242 ◽  
Author(s):  
Satoshi Usuki ◽  
Eri Kotani ◽  
Yumiko Kawakura ◽  
Masaki Sano ◽  
Yukitaka Katsura ◽  
...  
Keyword(s):  
Dna Synthesis ◽  
Estrous Cycle ◽  
Dna Polymerase ◽  
Preovulatory Follicle ◽  
Dna Polymerase Β ◽  
Dna Polymerase Α ◽  
Immature Rats ◽  
Α Activity

DNA polymerase α activity in ovaries of mature cycling rats during the normal estrous cycle changed in a cyclic manner with a peak at 1800 h in proestrus. Tokishakuyakusan (TS) in vivo did not affect the changes in DNA polymerase α and β activities during the estrous cycle. LH and FSH at 1000 or 1700 h in proestrus increased DNA polymerase α activity, but the DNA polymerase α activity induced by LH or FSH was not significantly affected by the addition of TS. DNA polymerase β activity did not change with LH, FSH or TS. In PMS-treated or -untreated immature rats, TS enhanced ovarian DNA polymerase α activity but had no significant effect on LH or FSH action. In ovaries, incubated in vitro, in untreated mature or immature rats, TS enhanced ovarian DNA polymerase α activity but had no significant effect on LH or FSH action. These results suggest that TS stimulates ovarian DNA polymerase α activity in relationship to DNA synthesis and does not affect the effect of LH or FSH on the activity by preovulatory follicle before and/or after the LH/FSH surge.

scholarly journals Highly Frequent Frameshift DNA Synthesis by Human DNA Polymerase μ

2001 ◽  
Vol 21 (23) ◽  
pp. 7995-8006 ◽  
Author(s):  
Yanbin Zhang ◽  
Xiaohua Wu ◽  
Fenghua Yuan ◽  
Zhongwen Xie ◽  
Zhigang Wang
Keyword(s):  
Dna Synthesis ◽  
Dna Polymerase ◽  
Somatic Hypermutation ◽  
Biochemical Properties ◽  
Nonhomologous End Joining ◽  
End Joining ◽  
Synthesis Mechanism ◽  
Biochemical Analyses

ABSTRACT DNA polymerase μ (Polμ) is a newly identified member of the polymerase X family. The biological function of Polμ is not known, although it has been speculated that human Polμ may be a somatic hypermutation polymerase. To help understand the in vivo function of human Polμ, we have performed in vitro biochemical analyses of the purified polymerase. Unlike any other DNA polymerases studied thus far, human Polμ catalyzed frameshift DNA synthesis with an unprecedentedly high frequency. In the sequence contexts examined, −1 deletion occurred as the predominant DNA synthesis mechanism opposite the single-nucleotide repeat sequences AA, GG, TT, and CC in the template. Thus, the fidelity of DNA synthesis by human Polμ was largely dictated by the sequence context. Human Polμ was able to efficiently extend mismatched bases mainly by a frameshift synthesis mechanism. With the primer ends, containing up to four mismatches, examined, human Polμ effectively realigned the primer to achieve annealing with a microhomology region in the template several nucleotides downstream. As a result, human Polμ promoted microhomology search and microhomology pairing between the primer and the template strands of DNA. These results show that human Polμ is much more prone to cause frameshift mutations than base substitutions. The biochemical properties of human Polμ suggest a function in nonhomologous end joining and V(D)J recombination through its microhomology searching and pairing activities but do not support a function in somatic hypermutation.

scholarly journals Murine cytomegalovirus resistant to antivirals has genetic correlates with human cytomegalovirus

2005 ◽  
Vol 86 (8) ◽  
pp. 2141-2151 ◽  
Author(s):  
G. M. Scott ◽  
H.-L. Ng ◽  
C. J. Morton ◽  
M. W. Parker ◽  
W. D. Rawlinson
Keyword(s):  
Protein Kinase ◽  
Dna Polymerase ◽  
Human Cytomegalovirus ◽  
Murine Cytomegalovirus ◽  
Cross Resistance ◽  
Antiviral Resistance ◽  
Three Dimensional Model ◽  
Mcmv Infection

Human cytomegalovirus (HCMV) resistance to antivirals is a significant clinical problem. Murine cytomegalovirus (MCMV) infection of mice is a well-described animal model for in vivo studies of CMV pathogenesis, although the mechanisms of MCMV antiviral susceptibility need elucidation. Mutants resistant to nucleoside analogues aciclovir, adefovir, cidofovir, ganciclovir, penciclovir and valaciclovir, and the pyrophosphate analogue foscarnet were generated by in vitro passage of MCMV (Smith) in increasing concentrations of antiviral. All MCMV antiviral resistant mutants contained DNA polymerase mutations identical or similar to HCMV DNA polymerase mutations known to confer antiviral resistance. Mapping of the mutations onto an MCMV DNA polymerase three-dimensional model generated using the Thermococcus gorgonarius Tgo polymerase crystal structure showed that the DNA polymerase mutations potentially confer resistance through changes in regions surrounding a catalytic aspartate triad. The ganciclovir-, penciclovir- and valaciclovir-resistant isolates also contained mutations within MCMV M97 identical or similar to recognized GCV-resistant mutations of HCMV UL97 protein kinase, and demonstrated cross-resistance to antivirals of the same class. This strongly suggests that MCMV M97 has a similar role to HCMV UL97 in the phosphorylation of nucleoside analogue antivirals. All MCMV mutants demonstrated replication-impaired phenotypes, with the lowest titre and plaque size observed for isolates containing mutations in both DNA polymerase and M97. These findings indicate DNA polymerase and protein kinase regions of potential importance for antiviral susceptibility and replication. The similarities between MCMV and HCMV mutations that arise under antiviral selective pressure increase the utility of MCMV as a model for in vivo studies of CMV antiviral resistance.

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