Arabinosylnucleoside 5′-Triphosphate Inhibits DNA Primase of Calf Thymus1

摘要

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 synthesis is more sensitive to araCTP than DNA-primed DNA synthesis (Yoshida, S.,et al. (1983)Biochim. Biophys. Acta741, 348–357). Here we measured DNA primase activity using poly(dT) template or M13 bacteriophage single-stranded DNA template and primer RNA synthesis was coupled to the reaction byEscherichia coliDNA polymerase I Klenow fragment. By this method, the primer RNA synthesis can be measured independently of the associating DNA polymeraseα. Using poly(dT) template, it was found that arabinosyladenine 5'-triphosphate (araATP) strongly inhibited DNA primase in competition with rATP. The apparentK1for araATP was 21μM and the ratio ofK1/Km(for rATP) was as low as 0.015. With poly(dI, dT) or M13 DNA, it was shown that araCTP also inhibited DNA primase in the similar manner. Product analysis using α-32PrATP 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 c