Journal of Virology, July 2001, p. 6392-6401, Vol. 75, No. 14
0022-538X/01/$04.00+0 DOI: 10.1128/JVI.75.14.6392-6401.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
-Primase Complex Is Not
Significantly Altered by Changes in the Concentration of
Ribonucleotides

Department of Biochemistry, Tufts University School of Medicine, Boston, Massachusetts 02111
Received 22 February 2001/Accepted 18 April 2001
The simian virus 40 (SV40) in vitro replication system was
previously used to demonstrate that the human polymerase (Pol)
-primase complex preferentially initiates DNA synthesis at
pyrimidine-rich trinucleotide sequences. However, it has been
reported that under certain conditions, nucleoside triphosphate (NTP)
concentrations play a critical role in determining where eukaryotic
primase initiates synthesis. Therefore, we have examined whether
increased NTP concentrations alter the template locations at which SV40
replication is initiated. Our studies demonstrate that elevated
ribonucleotide concentrations do not significantly alter which template
sequences serve as initiation sites. Of considerable interest, the
sequences that serve as initiation sites in the SV40 system are similar
to those that serve as initiation sites for prokaryotic primases. It is
also demonstrated that regardless of the concentration of
ribonucleotides present in the reactions, DNA synthesis initiated
outside of the core origin. These studies provide additional evidence
that the Pol
-primase complex can initiate DNA synthesis only after
a considerable amount of single-stranded DNA is generated.
Present address: Millennium Pharmaceutical, Inc., Cambridge, MA 02139.
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