scholarly journals DNA Synthesis on a Double-stranded DNA Template by the T4 Bacteriophage DNA Polymerase and the T4 Gene 32 DNA Unwinding Protein

1974 ◽  
Vol 249 (17) ◽  
pp. 5668-5676
Author(s):  
Nancy G. Nossal
Keyword(s):  
Dna Synthesis ◽  
Dna Polymerase ◽  
Dna Unwinding ◽  
Double Stranded Dna ◽  
T4 Bacteriophage ◽  
Gene 32 ◽  
Dna Template

scholarly journals Properties of mitochondrial DNA polymerase in mitochondrial DNA synthesis in yeast.

1995 ◽  
Vol 42 (3) ◽  
pp. 317-324 ◽  
Author(s):  
T K Biswas ◽  
P Sengupta ◽  
R Green ◽  
P Hakim ◽  
B Biswas ◽  
...  
Keyword(s):  
Mitochondrial Dna ◽  
Dna Synthesis ◽  
Dna Polymerase ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Mt Dna ◽  
Double Stranded Dna ◽  
Dna Template ◽  
Mitochondrial Dna Polymerase

Mitochondrial DNA polymerase from Saccharomyces cerevisiae, purified 3500 fold, was separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis into three polypeptides. The major 150 kDa polypeptide was probably the catalytic subunit of the mitochondrial (mt) DNA polymerase and the other two polypeptides could be either proteolytic cleavage products of the polymerase, other subunits of the enzyme or protein contaminants. The mtDNA polymerase preferred an A+T-rich DNA template and did not require any RNA primer for DNA synthesis, at least under in vitro reaction conditions. It showed higher processivity on a double-stranded linear DNA template than on a single-stranded circular DNA template, and was capable of synthesizing at least about 1200 nucleotide primer-extended products without any major pause on a double-stranded DNA template.

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 CMG–Pol epsilon dynamics suggests a mechanism for the establishment of leading-strand synthesis in the eukaryotic replisome

2017 ◽  
Vol 114 (16) ◽  
pp. 4141-4146 ◽  
Author(s):  
Jin Chuan Zhou ◽  
Agnieszka Janska ◽  
Panchali Goswami ◽  
Ludovic Renault ◽  
Ferdos Abid Ali ◽  
...  
Keyword(s):  
Dna Synthesis ◽  
Single Particle ◽  
Replication Fork ◽  
Genome Duplication ◽  
Chromosome Replication ◽  
Dna Unwinding ◽  
Conformational Switch ◽  
Leading Strand ◽  
Dna Template

The replisome unwinds and synthesizes DNA for genome duplication. In eukaryotes, the Cdc45–MCM–GINS (CMG) helicase and the leading-strand polymerase, Pol epsilon, form a stable assembly. The mechanism for coupling DNA unwinding with synthesis is starting to be elucidated, however the architecture and dynamics of the replication fork remain only partially understood, preventing a molecular understanding of chromosome replication. To address this issue, we conducted a systematic single-particle EM study on multiple permutations of the reconstituted CMG–Pol epsilon assembly. Pol epsilon contains two flexibly tethered lobes. The noncatalytic lobe is anchored to the motor of the helicase, whereas the polymerization domain extends toward the side of the helicase. We observe two alternate configurations of the DNA synthesis domain in the CMG-bound Pol epsilon. We propose that this conformational switch might control DNA template engagement and release, modulating replisome progression.

Prokaryotic DNA replication mechanisms

1987 ◽  
Vol 317 (1187) ◽  
pp. 395-420 ◽  
Keyword(s):  
Dna Replication ◽  
Dna Polymerase ◽  
Replication Fork ◽  
Kinetic Studies ◽  
Dna Helicase ◽  
Gene 32 ◽  
Dna Template ◽  
Lagging Strand

The three different prokaryotic replication systems that have been most extensively studied use the same basic components for moving a DNA replication fork, even though the individual proteins are different and lack extensive amino acid sequence homology. In the T4 bacteriophage system, the components of the DNA replication complex can be grouped into functional classes as follows: DNA polymerase (gene 43 protein), helix-destabilizing protein (gene 32 protein), polymerase accessory proteins (gene 44/62 and 45 proteins), and primosome proteins (gene 41 DNA helicase and gene 61 RNA primase). DNA synthesis in the in vitro system starts by covalent addition onto the 3'OH end at a random nick on a double-stranded DNA template and proceeds to generate a replication fork that moves at about the in vivo rate, and with approximately the in vivo base-pairing fidelity. DNA is synthesized at the fork in a continuous fashion on the leading strand and in a discontinuous fashion on the lagging strand (generating short Okazaki fragments with 5'-linked pppApCpXpYpZ pentaribonucleotide primers). Kinetic studies reveal that the DNA polymerase molecule on the lagging strand stays associated with the fork as it moves. Therefore the DNA template on the lagging strand must be folded so that the stop site for the synthesis of one Okazaki fragment is adjacent to the start site for the next such fragment, allowing the polymerase and other replication proteins on the lagging strand to recycle.

scholarly journals Structural basis of DNA synthesis opposite 8-oxoguanine by human PrimPol primase-polymerase

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Olga Rechkoblit ◽  
Robert E. Johnson ◽  
Yogesh K. Gupta ◽  
Louise Prakash ◽  
Satya Prakash ◽  
...  
Keyword(s):  
Dna Synthesis ◽  
Dna Polymerase ◽  
Thermodynamic Stability ◽  
Active Site ◽  
Base Pair ◽  
Translesion Synthesis ◽  
Human Cells ◽  
Structural Basis ◽  
Human Dna ◽  
Dna Template

AbstractPrimPol is a human DNA polymerase-primase that localizes to mitochondria and nucleus and bypasses the major oxidative lesion 7,8-dihydro-8-oxoguanine (oxoG) via translesion synthesis, in mostly error-free manner. We present structures of PrimPol insertion complexes with a DNA template-primer and correct dCTP or erroneous dATP opposite the lesion, as well as extension complexes with C or A as a 3′−terminal primer base. We show that during the insertion of C and extension from it, the active site is unperturbed, reflecting the readiness of PrimPol to accommodate oxoG(anti). The misinsertion of A opposite oxoG(syn) also does not alter the active site, and is likely less favorable due to lower thermodynamic stability of the oxoG(syn)•A base-pair. During the extension step, oxoG(syn) induces an opening of its base-pair with A or misalignment of the 3′-A primer terminus. Together, the structures show how PrimPol accurately synthesizes DNA opposite oxidatively damaged DNA in human cells.

scholarly journals Mechanism for priming DNA synthesis by yeast DNA Polymerase α

eLife ◽  
2013 ◽  
Vol 2 ◽  
Author(s):  
Rajika L Perera ◽  
Rubben Torella ◽  
Sebastian Klinge ◽  
Mairi L Kilkenny ◽  
Joseph D Maman ◽  
...  
Keyword(s):  
Dna Synthesis ◽  
Dna Polymerase ◽  
Catalytic Core ◽  
Dna Polymerase Α ◽  
Dna Oligonucleotides ◽  
Computational Data ◽  
Dna Template ◽  
Lagging Strand ◽  
Primase Complex ◽  
Nucleotide Polymerization

The DNA Polymerase α (Pol α)/primase complex initiates DNA synthesis in eukaryotic replication. In the complex, Pol α and primase cooperate in the production of RNA-DNA oligonucleotides that prime synthesis of new DNA. Here we report crystal structures of the catalytic core of yeast Pol α in unliganded form, bound to an RNA primer/DNA template and extending an RNA primer with deoxynucleotides. We combine the structural analysis with biochemical and computational data to demonstrate that Pol α specifically recognizes the A-form RNA/DNA helix and that the ensuing synthesis of B-form DNA terminates primer synthesis. The spontaneous release of the completed RNA-DNA primer by the Pol α/primase complex simplifies current models of primer transfer to leading- and lagging strand polymerases. The proposed mechanism of nucleotide polymerization by Pol α might contribute to genomic stability by limiting the amount of inaccurate DNA to be corrected at the start of each Okazaki fragment.

Stimulation of T4 bacteriophage DNA polymerase by the protein product of T4 gene 32

1971 ◽  
Vol 62 (1) ◽  
pp. 39-52 ◽  
Author(s):  
Joel A. Huberman ◽  
Arthur Kornberg ◽  
Bruce M. Alberts
Keyword(s):  
Dna Polymerase ◽  
Protein Product ◽  
T4 Bacteriophage ◽  
Gene 32 ◽  
Stimulation Of

scholarly journals Biochemical aspects of cardiac muscle differentiation. Determination of deoxyribonucleic acid template availability and 3′-hydroxyl termini in nuclei and chromatin by using exogenous deoxyribonucleic acid polymerases

1978 ◽  
Vol 171 (2) ◽  
pp. 289-298 ◽  
Author(s):  
William C. Claycomb
Keyword(s):  
Dna Synthesis ◽  
Cardiac Muscle ◽  
Dna Polymerase ◽  
Deoxyribonucleic Acid ◽  
Dna Polymerases ◽  
Dna Polymerase Α ◽  
Dna Template ◽  
Hydroxyl Termini ◽  
Density Shift

Experiments were designed to determine whether DNA synthesis ceases in terminally differentiating cardiac muscle of the rat because the activity of the putative replicative DNA polymerase (DNA polymerase α) is lost or whether the activity of this enzyme is lost because DNA synthesis ceases. DNA-template availability and 3′-hydroxyl termini in nuclei and chromatin, isolated from cardiac muscle at various times during the developmental period in which DNA synthesis and the activity of DNA polymerase α are decreasing, were measured by using Escherichia coli DNA polymerase I, Micrococcus luteus DNA polymerase and DNA polymerase α under optimal conditions. Density-shift experiments with bromodeoxyuridine triphosphate and isopycnic analysis indicate that DNA chains being replicated semi-conservatively in vivo continue to be elongated in isolated nuclei by exogenous DNA polymerases. DNA template and 3′-hydroxyl termini available to exogenously added DNA polymerases do not change as cardiac muscle differentiates and the rate of DNA synthesis decreases and ceases in vivo. Template availability and 3′-hydroxyl termini are also not changed in nuclei isolated from cardiac muscle in which DNA synthesis had been inhibited by administration of isoproterenol and theophylline to newborn rats. DNA-template availability and 3′-hydroxyl termini, however, were substantially increased in nuclei and chromatin from cardiac muscle of adult rats. This increase is not due to elevated deoxyribonuclease activity in nuclei and chromatin of the adult. Electron microscopy indicates that this increase is also not due to dispersal of the chromatin or disruption of nuclear morphology. Density-shift experiments and isopycnic analysis of DNA from cardiac muscle of the adult show that it is more fragmented than DNA from cardiac-muscle cells that are, or have recently ceased, dividing. These studies indicate that DNA synthesis ceases in terminally differentiating cardiac muscle because the activity of a replicative DNA polymerase is lost, rather than the activity of this enzyme being lost because DNA synthesis ceases.

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