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

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.degree.C, 5 min) AS 65 fraction restored replication activity to the combination of micrococcal nuclease-treated AS 40 and AS 65 fractions.