Arabinosylnucleoside 5'-Triphosphate Inhibits DNA Primase of Calf Thymus1

It has been shown that DNA primase activity is tightly associated with 10S DNA polymerase a from calf thymus and that the ribonucleotide-dependent DNA syn-thesis 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 pri-mase activity using poly(dT) template or Ml3 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 a. Using poly(dT) template, it was found that arabinosyladenine 5'-triphosphate (araATP) strongly inhibited DNA primase in competition with rATP. The apparent Ki for araATP was 21 //M and the ratio of KJKm (for rATP) was as low as 0.015. With poly(dl, dT) or Ml3 DNA, it was shown that araCTP also inhibited DNA primase in the similar manner. Product analysis using [a-"P]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 arabino-sylnucleotide inhibits the initiation as well as elongation of Okazaki fragments in mammalian cells. DNA primase, an activity which synthesizes oligo- eukaryotes {1-12). With one exception (12), DNA nucleotide primer for DNA synthesis on unprimed primase was found in association with DNA poly-DNA template, has been found in a variety of merase a. These two activities were co-purified through a series of column chromatographies. Se-, _..!., _..... „ paration was achieved under drastic condition such 1 This work was supported by a Grant-in-Aid for Cancer