Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 1986 Jul;83(13):4612-6.
doi: 10.1073/pnas.83.13.4612.

Simian virus 40 DNA replication in vitro: study of events preceding elongation of chains

C R Wobbe, F B Dean, Y Murakami, L Weissbach, J Hurwitz

Simian virus 40 DNA replication in vitro: study of events preceding elongation of chains

C R Wobbe et al. Proc Natl Acad Sci U S A. 1986 Jul.

Abstract

We have evidence for the formation of a stable preelongation complex during the replication of simian virus 40 (SV40) origin containing DNA (ori+ DNA) in vitro. Preincubation of ori+ DNA with HeLa cytosolic extracts and SV40-encoded large tumor antigen (T antigen) in the absence of deoxynucleoside triphosphates eliminates a lag that normally precedes replication. This effect requires ATP and is inhibited by RNase A; subsequent elongation is inhibited by aphidicolin but not by RNase A. A T antigen and SV40 origin-dependent complex can be isolated by gel-filtration chromatography of preincubation reaction mixtures. In both cases, the products formed by replication after complex formation resemble those formed during in vitro replication reactions described previously. HeLa cytosolic extract was separated into two ammonium sulfate fractions: a 0-40% fraction (AS 40) that shows low levels of DNA synthesis and a 40-65% fraction (AS 65) that is inactive by itself but stimulates synthesis when added to the AS 40 fraction. DNA synthesis by these combined fractions has the same requirements as crude extract, occurs in two stages as described above, and is sensitive to RNase A. Pretreatment of both fractions with micrococcal nuclease eliminated replication activity, whereas the combination of a pretreated fraction (either AS 40 or 65) with an untreated fraction was active. A heat-inactivated (55 degrees C, 5 min) AS 65 fraction restored replication activity to the combination of micrococcal nuclease-treated AS 40 and AS 65 fractions.

PubMed Disclaimer

References

    1. Proc Natl Acad Sci U S A. 1985 Jun;82(12):3954-8 - PubMed
    1. CRC Crit Rev Biochem. 1984;17(2):153-215 - PubMed
    1. EMBO J. 1985 Nov;4(11):2933-9 - PubMed
    1. Proc Natl Acad Sci U S A. 1986 May;83(9):2869-73 - PubMed
    1. Mol Cell Biol. 1985 Aug;5(8):2051-60 - PubMed

Publication types