Effect of ribonucleotides on substrate availability for DNA polymerase assays in cytoplasmic fractions of rat intestinal mucosa

1977 ◽  
Vol 55 (4) ◽  
pp. 489-492
Author(s):  
S. H. Zbarsky ◽  
S. Chevalier
Keyword(s):  
Dna Synthesis ◽  
Dna Polymerase ◽  
Intestinal Mucosa ◽  
Inorganic Phosphate ◽  
Enzymatic Degradation ◽  
Polymerase Activity ◽  
Subcellular Fractions ◽  
Substrate Availability ◽  
Thymidine Triphosphate ◽  
Deoxyribonucleoside Triphosphate

The deoxyribonucleoside triphosphate substrates for DNA synthesis were hydrolysed during the DNA polymerase (EC 2.7.7.7) assay with cytoplasmic subcellular fractions of rat intestinal mucosa. Presumably because of phosphatase (EC 3.1.3.2) activity in these fractions, inorganic phosphate was liberated from the nucleotides, and radioactive thymidine triphosphate was shown to be degraded to thymidine di- and mono-phosphate, thymidine, and thymine. Addition of ATP to the postmicrosomal supernatant increased its DNA polymerase activity by sparing the deoxyribonucleotide precursors from enzymatic degradation.

Terminal deoxyribonucleotidyltransferase from nuclei of rat intestinal mucosa

1970 ◽  
Vol 48 (5) ◽  
pp. 537-540 ◽  
Author(s):  
F. Y. T. Leung ◽  
S. H. Zbarsky
Keyword(s):  
Dna Polymerase ◽  
Snake Venom ◽  
Intestinal Mucosa ◽  
Deae Cellulose ◽  
Nuclear Extract ◽  
Polymerase Activity ◽  
Snake Venom Phosphodiesterase ◽  
Deoxyribonucleoside Triphosphate

A terminal deoxyribonucleotidyltransferase has been isolated from extracts of nuclei of rat intestinal mucosa. The enzyme was obtained in partially purified form by rechromatography of material with DNA polymerase activity isolated previously by chromatography of the nuclear extract on DEAE-cellulose. The enzyme, which required heat-denatured DNA as a primer, catalyzed the incorporation of radioactivity from a single labelled deoxyribonucleoside triphosphate into the DNA product. The incorporation was inhibited up to 70% in the presence of all four complementary deoxyribonucleoside triphosphates. Evidence has been presented based on the release of radioactivity from the DNA product during its hydrolysis by snake venom phosphodiesterase which indicates that the enzyme catalyzes the addition of deoxyribonucleotides to the 3′-OH terminus of heat-denatured DNA. The properties of the enzyme are those of a terminal deoxyribonucleotidyltransferase as distinct from the replicative transferase, DNA polymerase.

scholarly journals Heterotrimeric PCNA Increases the Activity and Fidelity of Dbh, a Y-family Translesion DNA Polymerase Prone to Creating Single-Base Deletion Mutations

2020 ◽  
Author(s):  
Yifeng Wu ◽  
William Jaremko ◽  
Ryan C. Wilson ◽  
Janice D. Pata
Keyword(s):  
Dna Synthesis ◽  
Dna Polymerase ◽  
Polymerase Activity ◽  
List Type ◽  
Single Base ◽  
Deletion Mutations ◽  
Single Base Deletion ◽  
Translesion Dna ◽  
Y Family

AbstractDbh is a Y-family translesion DNA polymerase from Sulfolobus acidocaldarius, an archaeal species that grows in harsh environmental conditions. Biochemically, Dbh displays a distinctive mutational profile, creating single-base deletion mutations at extraordinarily high frequencies (up to 50%) in specific repeat sequences. In cells, however, Dbh does not appear to contribute significantly to spontaneous frameshifts in these same sequence contexts. This suggests that either the error-prone DNA synthesis activity of Dbh is reduced in vivo and/or Dbh is restricted from replicating these sequences. Here, we test the hypothesis that the propensity for Dbh to make single base deletion mutations is reduced through interaction with the S. acidocaldarius heterotrimeric sliding clamp processivity factor, PCNA-123. We first confirm that Dbh physically interacts with PCNA-123, with the interaction requiring both the PCNA-1 subunit and the C-terminal 10 amino acids of Dbh, which contain a predicted PCNA-interaction peptide (PIP) motif. This interaction stimulates the polymerase activity of Dbh, even on short, linear primer-template DNA by increasing the rate of nucleotide incorporation. This stimulation requires an intact PCNA-123 heterotrimer and a DNA duplex length of at least 18 basepairs, the minimal length predicted from structural data to bind to both the polymerase and the clamp. Finally, we find that PCNA-123 increases the fidelity of Dbh on a single-base deletion hotspot sequence 3-fold by promoting an increase in the rate of correct, but not incorrect, nucleotide addition and propose that PCNA-123 induces Dbh to adopt a more active conformation that is less prone to creating deletions during DNA synthesis.HighlightsPCNA increases the fidelity of Dbh polymerase on a deletion-hotspot sequence.The interaction stimulates incorporation of the correct, but not incorrect, nucleotide.A minimal duplex length of 18 bp is required for PCNA to stimulate polymerase activity.Structural modeling suggests that PCNA induces a conformational change in Dbh.

scholarly journals REPAIR DNA SYNTHESIS IN DIFFERENTIATED EMBRYONIC MUSCLE CELLS

1972 ◽  
Vol 52 (3) ◽  
pp. 589-597 ◽  
Author(s):  
Frank E. Stockdale ◽  
Michael C. O'neill
Keyword(s):  
Skeletal Muscle ◽  
Dna Synthesis ◽  
Dna Polymerase ◽  
Uv Irradiation ◽  
Life Time ◽  
Muscle Cells ◽  
Polymerase Activity ◽  
Repair Synthesis ◽  
Entire Life ◽  
Embryonic Muscle

The differentiation of embryonic skeletal muscle cells is closely coupled with the cessation of normal DNA replication. Once these cells begin to differentiate, they normally never undergo semiconservative replication of DNA during the entire life time of the muscle cell. Cessation of DNA synthesis has been shown to be accompanied by a loss of 80–90% of the replicative DNA polymerase activity of these cells. Despite this loss the studies reported here demonstrate that muscle cells retain the ability to synthesize DNA of a repair type after UV irradiation. These results suggest that the control exercised over semiconservative DNA synthesis during differentiation of these cells does not extend to repair synthesis after UV irradiation.

Effect of benzo[a]pyrene on DNA synthesis and DNA polymerase activity of rat liver nuclei

1985 ◽  
Vol 34 (6) ◽  
pp. 755-762 ◽  
Author(s):  
Inés Salazar ◽  
Laura Tarragó-Litvak ◽  
Simón Litvak ◽  
Lionel Gil
Keyword(s):  
Dna Synthesis ◽  
Dna Polymerase ◽  
Rat Liver ◽  
Polymerase Activity ◽  
Rat Liver Nuclei ◽  
Liver Nuclei

scholarly journals Poly(adenosine diphosphate ribose) polymerase activity and nicotinamide adenine dinucleotide in differentiating cardiac muscle

1976 ◽  
Vol 154 (2) ◽  
pp. 387-393 ◽  
Author(s):  
W C. Claycomb
Keyword(s):  
Cyclic Amp ◽  
Dna Synthesis ◽  
Cardiac Muscle ◽  
Dna Polymerase ◽  
Specific Activity ◽  
Polymerase Activity ◽  
Neonatal Rat ◽  
Dibutyryl Cyclic Amp ◽  
Fresh Tissue ◽  
Dna Polymerase Α

Poly(ADP-ribose) polymerase activity in nuclei isolated from differentiating cardiac muscle of the rat has been characterized and its activity measured during development. Optimum enzyme activity is observed at pH 8.5. Poly(ADP-ribose) polymerase is inhibited by ATP, thymidine, nicotinamide, theophylline, 3-isobutyl-1-methylxanthine and caffeine and stimulated by actinomycin D. The activity measured under optimal assay conditions increases during differentiation of cardiac muscle and is inversely related to the rate of DNA synthesis and to the activities of DNA polymerase α and thymidine kinase. When DNA synthesis and the activity of DNA polymerase α are inhibited in cardiac muscle of the 1-day-old neonatal rat by dibutyryl cyclic AMP or isoproterenol, the specific activity of poly(ADP-ribose) polymerase measured in isolated nuclei is increased. The concentration of NAD+ in cardiac muscle increases during postnatal development. In the adult compared with the 1-day-old neonatal rat the concentration of NAD+ relative to fresh tissue weight, DNA or protein increased 1.7-fold, 5.2-fold or 1.4-fold respectively. The concentration of NAD+ in cardiac muscle of the 1-day-old neonatal rat can be increased by approx. 20% by dibutyryl cyclic AMP. These data suggest that NAD+ and poly(ADP-ribose) polymerase may be involved with the repression of DNA synthesis and cell proliferation in differentiating cardiac muscle.

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