scholarly journals Fidelity of DNA synthesis by the Thermococcus litoralis DNA polymerase—an extremely heat stable enzyme with proofreading activity

1991 ◽  
Vol 19 (18) ◽  
pp. 4967-4973 ◽  
Author(s):  
P. Mattila ◽  
J. Korpela ◽  
T. Tenkanen ◽  
K. Pitkämem
Keyword(s):  
Dna Synthesis ◽  
Dna Polymerase ◽  
Dna Sequences ◽  
Total Concentration ◽  
Dna Amplification ◽  
Base Substitution ◽  
Thermococcus Litoralis ◽  
Heat Stable ◽  
Original Target

Abstract We demonstrate that the DNA polymerase isolated from Thermococcus litoralis (VentTM DNA polymerase) is the first thermostable DNA polymerase reported having a 3′—5′ proofreading exonuclease activity. This facilitates a highly accurate DNA synthesis in vitro by the polymerase. Mutational frequencies observed in the base substitution fidelity assays were in the range of 30×10−6. These values were 5–10 times lower compared to other thermostable DNA polymerases lacking the proofreading activity. All classes of DNA polymerase errors (transitions, transversions, frameshift mutations) were assayed using the forward mutational assay (1). The mutation frequencies of Thermococcus litoralis DNA polymerase varied between 15−35×10−4 being 2 – 4 times lower than the respective values obtained using enzymes without proofreading activity. We also noticed that the fidelity of the DNA polymerase from Thermococcus litoralis responds to changes in dNTP concentration, units of enzyme used per one reaction and the concentration of MgSO4 relative to the total concentration of dNTPs present in the reaction. The high fidelity DNA synthesis In vitro by Thermococcus litoralis DNA polymerase provides good possibilities for maintaining the genetic information of original target DNA sequences intact in the DNA amplification applications.

scholarly journals Involvement of deoxyribonucleic acid polymerase β in nuclear deoxyribonucleic acid synthesis

1978 ◽  
Vol 173 (1) ◽  
pp. 309-314 ◽  
Author(s):  
T R Butt ◽  
W M Wood ◽  
E L McKay ◽  
R L P Adams
Keyword(s):  
Dna Synthesis ◽  
Dna Polymerase ◽  
Deoxyribonucleic Acid ◽  
Nuclear Dna ◽  
Dna Polymerase Beta ◽  
Cell Nuclei ◽  
High Molecular Weight Dna ◽  
Dna Polymerase Alpha ◽  
Polymerase Beta

The effects on DNA synthesis in vitro in mouse L929-cell nuclei of differential extraction of DNA polymerases alpha and beta were studied. Removal of all measurable DNA polymerase alpha and 20% of DNA polymerase beta leads to a 40% fall in the replicative DNA synthesis. Removal of 70% of DNA polymerase beta inhibits replicative synthesis by 80%. In all cases the nuclear DNA synthesis is sensitive to N-ethylmaleimide and aCTP (arabinosylcytosine triphosphate), though less so than DNA polymerase alpha. Addition of deoxyribonuclease I to the nuclear incubation leads to synthesis of high-molecular-weight DNA in a repair reaction. This occurs equally in nuclei from non-growing or S-phase cells. The former nuclei lack DNA polymerase alpha and the reaction reflects the sensitivity of DNA polymerase beta to inhibiton by N-ethylmaleimide and aCTP.

Oligo(A)-stimulated Tetrahymena rDNA synthesis in vitro

1981 ◽  
Vol 59 (6) ◽  
pp. 396-403 ◽  
Author(s):  
Peter R. Ganz ◽  
Gyorgy B. Kiss ◽  
Ronald E. Pearlman
Keyword(s):  
Rna Polymerase ◽  
Dna Synthesis ◽  
Dna Polymerase ◽  
Ribosomal Rna ◽  
Replication Proteins ◽  
General Applicability ◽  
In Vitro Synthesis ◽  
Restriction Fragments ◽  
Double Stranded Dna

The synthesis of Tetrahymena rDNA has been examined using purified DNA polymerase and partially purified preparations of homologous replication enzymes (fraction IV). DNA synthesis with purified DNA polymerase alone was less than that with fraction IV enzymes. This suggested that there were additional factors in fraction IV other than DNA polymerase which contributed to or enhanced rDNA synthesis in vitro. Neither hybridization of rDNA with Tetrahymena ribosomal RNA nor preincubation of rDNA with homologous or heterologous RNA polymerase served to stimulate in vitro synthesis by fraction IV enzymes. However, when rDNA was hybridized with oligoriboadenylate, DNA synthesis using fraction IV was stimulated approximately 4- to 4.5-fold over 150 min of incubation, relative to a similarly treated but unhybridized rDNA control. Using oligoriboadenylate-hybridized EcoR1 and HindIII restriction fragments of rDNA to localize the synthesis most of the in vitro synthesis occurred within a 2.4 × 106 Mr fragment encompassing the centre of the rDNA molecule. The approach of hybridizing a synthetic homooligoribonucleotide primer to double-stranded DNA should prove to be of general applicability in designing similar template–primers in other systems for the purpose of isolating replication proteins.

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.

scholarly journals Minidumbbell structures formed by ATTCT pentanucleotide repeats in spinocerebellar ataxia type 10

2020 ◽  
Author(s):  
Pei Guo ◽  
Sik Lok Lam
Keyword(s):  
Spinocerebellar Ataxia ◽  
Dna Polymerase ◽  
Dna Sequences ◽  
Solution Structure ◽  
Large Scale ◽  
Genetic Disorder ◽  
Spinocerebellar Ataxia Type ◽  
Klenow Fragment ◽  
Repeat Expansions

Abstract Spinocerebellar ataxia type 10 (SCA10) is a progressive genetic disorder caused by ATTCT pentanucleotide repeat expansions in intron 9 of the ATXN10 gene. ATTCT repeats have been reported to form unwound secondary structures which are likely linked to large-scale repeat expansions. In this study, we performed high-resolution nuclear magnetic resonance spectroscopic investigations on DNA sequences containing two to five ATTCT repeats. Strikingly, we found the first two repeats of all these sequences well folded into highly compact minidumbbell (MDB) structures. The 3D solution structure of the sequence containing two ATTCT repeats was successfully determined, revealing the MDB comprises a regular TTCTA and a quasi TTCT/A pentaloops with extensive stabilizing loop-loop interactions. We further carried out in vitro primer extension assays to examine if the MDB formed in the primer could escape from the proofreading function of DNA polymerase. Results showed that when the MDB was formed at 5-bp or farther away from the priming site, it was able to escape from the proofreading by Klenow fragment of DNA polymerase I and thus retained in the primer. The intriguing structural findings bring about new insights into the origin of genetic instability in SCA10.

scholarly journals Initiation of simian virus 40 DNA replication in vitro: aphidicolin causes accumulation of early-replicating intermediates and allows determination of the initial direction of DNA synthesis.

1986 ◽  
Vol 6 (11) ◽  
pp. 3815-3825 ◽  
Author(s):  
R S Decker ◽  
M Yamaguchi ◽  
R Possenti ◽  
M L DePamphilis
Keyword(s):  
Dna Replication ◽  
Dna Synthesis ◽  
Dna Polymerase ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
T Antigen ◽  
Early Gene ◽  
Initial Direction ◽  
Dna Polymerase Alpha

Aphidicolin, a specific inhibitor of DNA polymerase alpha, provided a novel method for distinguishing between initiation of DNA synthesis at the simian virus 40 (SV40) origin of replication (ori) and continuation of replication beyond ori. In the presence of sufficient aphidicolin to inhibit total DNA synthesis by 50%, initiation of DNA replication in SV40 chromosomes or ori-containing plasmids continued in vitro, whereas DNA synthesis in the bulk of SV40 replicative intermediate DNA (RI) that had initiated replication in vivo was rapidly inhibited. This resulted in accumulation of early RI in which most nascent DNA was localized within a 600- to 700-base-pair region centered at ori. Accumulation of early RI was observed only under conditions that permitted initiation of SV40 ori-dependent, T-antigen-dependent DNA replication and only when aphidicolin was added to the in vitro system. Increasing aphidicolin concentrations revealed that DNA synthesis in the ori region was not completely resistant to aphidicolin but simply less sensitive than DNA synthesis at forks that were farther away. Since DNA synthesized in the presence of aphidicolin was concentrated in the 300 base pairs on the early gene side of ori, we conclude that the initial direction of DNA synthesis was the same as that of early mRNA synthesis, consistent with the model proposed by Hay and DePamphilis (Cell 28:767-779, 1982). The data were also consistent with initiation of the first DNA chains in ori by CV-1 cell DNA primase-DNA polymerase alpha. Synthesis of pppA/G(pN)6-8(pdN)21-23 chains on a single-stranded DNA template by a purified preparation of this enzyme was completely resistant to aphidicolin, and further incorporation of deoxynucleotide monophosphates was inhibited. Therefore, in the presence of aphidicolin, this enzyme could initiate RNA-primed DNA synthesis at ori first in the early gene direction and then in the late gene direction, but could not continue DNA synthesis for an extended distance.

A MAJOR ROLE FOR BACTERIOPHAGE T4 DNA POLYMERASE IN FRAMESHIFT MUTAGENESIS

Genetics ◽  
1983 ◽  
Vol 103 (3) ◽  
pp. 353-366
Author(s):  
Lynn S Ripley ◽  
Nadja B Shoemaker
Keyword(s):  
Dna Polymerase ◽  
Dna Sequences ◽  
Frameshift Mutation ◽  
Bacteriophage T4 ◽  
Base Substitution ◽  
Temperature Sensitive ◽  
Polymerase Gene ◽  
Frameshift Mutagenesis ◽  
Substitution Mutations ◽  
Polymerase Mutants

ABSTRACT T4 DNA polymerase strongly influences the frequency and specificity of frameshift mutagenesis. Fifteen of 19 temperature-sensitive alleles of the DNA polymerase gene substantially influenced the reversion frequencies of frameshift mutations measured in the T4 rII genes. Most polymerase mutants increased frameshift frequencies, but a few alleles (previously noted as antimutators for base substitution mutations) decreased the frequencies of certain frameshifts while increasing the frequencies of others. The various patterns of enhanced or decreased frameshift mutation frequencies suggest that T4 DNA polymerase is likely to play a variety of roles in the metabolic events leading to frameshift mutation. A detailed genetic study of the specificity of the mutator properties of three DNA polymerase alleles (tsL56, tsL98 and tsL88) demonstrated that each produces a distinctive frameshift spectrum. Differences in frameshift frequencies at similar DNA sequences within the rII genes, the influence of mutant polymerase alleles on these frequencies, and the presence or absence of the dinucleotide sequence associated with initiation of Okazaki pieces at the frameshift site has led us to suggest that the discontinuities associated with discontinuous DNA replication may contribute to spontaneous frameshift mutation frequencies in T4.

scholarly journals Detection of occult follicular lymphoma by specific DNA amplification

Blood ◽  
1988 ◽  
Vol 72 (5) ◽  
pp. 1822-1825 ◽  
Author(s):  
M Stetlet-Stevenson ◽  
M Raffeld ◽  
P Cohen ◽  
J Cossman
Keyword(s):  
Follicular Lymphoma ◽  
Dna Sequences ◽  
Genomic Dna ◽  
Restriction Analysis ◽  
Dna Amplification ◽  
Direct Application ◽  
Chain Reaction ◽  
Heat Stable ◽  
Valuable Adjunct ◽  
Polymerase Chain

Abstract To detect occult lymphoma, the polymerase chain reaction (PCR) technique was used to amplify joined bcl-2/JH DNA sequences at the juncture of the t(14:18) translocation in follicular lymphoma. Using the heat-stable DNA polymerase Taq and automated cycling of the reaction, we were able to detect as few as one to two copies of bcl- 2/JH. Under these conditions, PCR proved to be at least 10,000-fold more sensitive than either conventional flow cytometry or Southern blot restriction analysis. In addition, genomic DNA sequences of four lymphomas confirmed that the size of the amplified segment serves as a tumor marker. Direct application of PCR to patient staging revealed occult malignant lymphoma in tissue otherwise considered uninvolved by standard criteria. We conclude that the striking enhancement in diagnostic sensitivity attained by DNA amplification can serve as a valuable adjunct to the staging and clinical monitoring of follicular lymphoma.

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