Abstract
It has been shown that DNA primase activity is tightly associated with 10 S DNA polymerase α from calf thymus and that the ribonucleotide-dependent DNA synthesis is more sensitive to araCTP than DNA-primed DNA synthesis (Yoshida, S., et al. (1983) Biochim. Biophys. Acta 741, 348-357). Here we measured DNA primase activity using poly(dT) template or M 13 bacteriophage single-stranded DNA template and primer RNA synthesis was coupled to the reaction by Escherichia coli DNA polymerase I Klenow fragment. By this method, the primer RNA synthesis can be measured independently of the associating DNA polymerase α. Using poly(dT) template, it was found that arabinosyladenine 5'-triphosphate (araATP) strongly inhibited DNA primase in competition with rATP. The apparent K1 for araATP was 21 μM and the ratio of K1/Km (for rATP) was as low as 0.015. With poly(dI, dT) or M 13 DNA, it was shown that araCTP also inhibited DNA primase in the similar manner. Product analysis using [α-32P]rATP showed that araATP inhibited the elongation of primer RNA. However, it is not likely that arabinosyl nucleotides act as chain-terminators, since incubation of primer RNA with araATP did not abolish its priming activity. From these results, it is suggested that arabinosyl nucleotide inhibits the initiation as well as elongation of Okazaki fragments in mammalian cells.
