J. Biol. Chem., Vol. 255, Issue 20, 9552-9555, 10, 1980
Deoxyuridine triphosphate pools after polyoma virus infection
S Nilsson, P Reichard and L Skoog
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.
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