Abstract
The synthesis of polyoma DNA in virus-infected 3T6 mouse fibroblasts is discontinuous with the intermediate formation of short Okazaki fragments. Hydroxyurea, an inhibitor of the enzyme ribonucleotide reductase, inhibits polyoma DNA synthesis, as measured by incorporation of radioactive thymidine. In the inhibited state, almost all incorporation occurs into short fragments. We investigated to what extent formation of short DNA fragments might be the result of incorporation of deoxyuridine triphosphate (dUTP) into DNA, followed by excision and repair reactions. We devised a sensitive enzymatic method for measuring dUTP in cell extracts which allows the determination of the dUTP pool when this pool amounts to between 0.1 and 2% of the dTTP pool. No dUTP was detected in growing mouse fibroblasts. After infection with polyoma virus cell extracts contained 0.4% dUTP (of dTTP) at the peak of DNA synthesis. Addition of hydroxyurea at this point led to a disappearance of dUTP. We conclude that dUTP incorporation can contribute only minimally to the generation of short fragments during polyoma DNA synthesis.
Highlights
A uracil-containing polymer is synthesized frdoUmTP,dTTP, substitu residues tes can for dTTP thinis reaction
The cellular concentration of the nucleotide depends on the relative rates of the enzyme reaction leading to itssynthesis and those leading to itsremoval
The scheme shows that dUTPase is required for the biosynthesis of dTTP, one of the four building blocksof DNA. dUTP itself can act as such a building block by replacing dTTP [6,7,8,9, 12]
Summary
Nuclei from polyoma-infected cells have the potential to generate short DNA strands via uracil incorporation if supplied with a sufficiently large pool of dUTP [12]. For the determination of dUTP (from [14C]dATP)polymer wastreated withuracil glycosylase an amount of cell extract containing 50 or 100 pmol of dTTP was and alkali before the centrifugation step (Fig. 2 A ). The reaction was stopped with 5 pl of 6 M NaOH after presence of as little as 0.25 pmol of dUTP together with100 addition of 10 nmol of carrier commercial poly[d(A-T)] and incubated pmol of d T T P during thepolymerization step shifted tpheak further for 45 min at 37°C before centrifugation oann alkaline sucrose gradient.
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