Replication of vaccinia DNA in mouse L cells II. In vitro DNA synthesis in cytoplasmic extracts

Abstract

Cytoplasmic extracts prepared from vaccinia virus-infected L cells catalyzed the incorporation of labeled deoxynucleotide triphosphates into DNA which hybridized with vaccinia virus DNA. The incorporation of [ 3H]thymidine 5′ triphosphate ([ 3H]TTP) into DNA was shown to be dependent on the presence of all four deoxynucleoside triphosphates and incorporation was stimulated twofold by the addition of ATP, NAD, and ribonucleoside triphosphates. The incorporation of [ 3H]TTP in vitro was linear for 10 min and continued at a reduced rate for 30 min at 30°. The viral DNA synthesized in vitro was analyzed by sedimentation in alkaline-sucrose gradients and by isopycnic centrifugation in CsCl gradients. Alkaline-sucrose sedimentation analysis showed that replication of in vitro labeled DNA was discontinuous. Small fragments (∼10 S) were synthesized in vitro in 10–30 sec which appeared to elongate so that after 30 min of synthesis the in vitro synthesized molecules cosedimented with in vivo labeled viral DNA species of 10–70 S. No molecules of greater length than mature viral single-stranded DNA (Type 1, 70–72 S) were observed when cell extracts prepared 3 hr postinfection were employed. Replication of viral DNA in vitro was symmetrical. No evidence for circular or superhelical DNA duplex molecules was obtained when in vitro synthesized DNA was analyzed by equilibrium density centrifugation in CsCl containing ethidium bromide.