Initiation, elongation and pausing of in vitro DNA synthesis catalyzed by immunopurified yeast DNA primase: DNA polymerase complex.

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.

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Oligo(A)-stimulated Tetrahymena rDNA synthesis in vitro

Canadian Journal of Biochemistry ◽

10.1139/o81-055 ◽

1981 ◽

Vol 59 (6) ◽

pp. 396-403 ◽

Cited By ~ 2

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.

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

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.41.3.594 ◽

1997 ◽

Vol 41 (3) ◽

pp. 594-599 ◽

Cited By ~ 121

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.

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Stimulation of DNA replication by growth factor and hormones in Swiss 3T3 cells: Comparison of the rate of entry into S phase with in vitro DNA synthesis and DNA polymerase ? activity

Journal of Cellular Physiology ◽

10.1002/jcp.1041340107 ◽

1988 ◽

Vol 134 (1) ◽

pp. 57-66 ◽

Cited By ~ 3

Author(s):

Angela M. Otto ◽

Christina Smith ◽

Luis Jimenez De Asua

Keyword(s):

Dna Replication ◽

Growth Factor ◽

Dna Synthesis ◽

Dna Polymerase ◽

Polymerase Activity ◽

S Phase ◽

3T3 Cells ◽

Swiss 3T3 ◽

Stimulation Of

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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.

Molecular and Cellular Biology ◽

10.1128/mcb.6.11.3815 ◽

1986 ◽

Vol 6 (11) ◽

pp. 3815-3825 ◽

Cited By ~ 41

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.

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Primer release is the rate-limiting event in lagging-strand synthesis mediated by the T7 replisome

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1604894113 ◽

2016 ◽

Vol 113 (21) ◽

pp. 5916-5921 ◽

Cited By ~ 8

Author(s):

Alfredo J. Hernandez ◽

Seung-Joo Lee ◽

Charles C. Richardson

Keyword(s):

Dna Synthesis ◽

Dna Polymerase ◽

Fragment Length ◽

Dna Binding Protein ◽

Okazaki Fragment ◽

Dna Primase ◽

Dna Strands ◽

Leading Strand ◽

Rate Limiting ◽

Lagging Strand

DNA replication occurs semidiscontinuously due to the antiparallel DNA strands and polarity of enzymatic DNA synthesis. Although the leading strand is synthesized continuously, the lagging strand is synthesized in small segments designated Okazaki fragments. Lagging-strand synthesis is a complex event requiring repeated cycles of RNA primer synthesis, transfer to the lagging-strand polymerase, and extension effected by cooperation between DNA primase and the lagging-strand polymerase. We examined events controlling Okazaki fragment initiation using the bacteriophage T7 replication system. Primer utilization by T7 DNA polymerase is slower than primer formation. Slow primer release from DNA primase allows the polymerase to engage the complex and is followed by a slow primer handoff step. The T7 single-stranded DNA binding protein increases primer formation and extension efficiency but promotes limited rounds of primer extension. We present a model describing Okazaki fragment initiation, the regulation of fragment length, and their implications for coordinated leading- and lagging-strand DNA synthesis.

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