Abstract
An initial step in the replication of simian virus (SV40) DNA is the ATP-dependent formation of a double hexamer of the SV40 large tumor (T) antigen at the SV40 DNA replication origin. In the absence of DNA, T antigen assembled into hexamers in the presence of magnesium and ATP. Hexameric T antigen was stable and could be isolated by glycerol gradient centrifugation. The ATPase activities of hexameric and monomeric T antigen isolated from parallel glycerol gradients were identical. However, while monomeric T antigen was active in the ATP-dependent binding, untwisting, unwinding, and replication of SV40 origin-containing DNA, hexameric T antigen was inactive in these reactions. Isolated hexamers incubated at 37 degrees C in the presence of ATP remained intact, but dissociated into monomers when incubated at 37 degrees C in the absence of ATP. This dissociation restored the activity of these preparations in the DNA replication reaction, indicating that hexameric T antigen is not permanently inactivated but merely assembled into a nonproductive structure. We propose that the two hexamers of T antigen at the SV40 origin assemble around the DNA from monomer T antigen in solution. This complex untwists the DNA at the origin, melting specific DNA sequences. The resulting single-stranded regions may be utilized by the T antigen helicase activity to initiate DNA unwinding bidirectionally from the origin.
Highlights
An initial step in the replication of simian virus to the SV40 replication origin
While monomeric T antigen was active in the ATP-dependent binding, untwisting, unwinding, and replication of SV40 origin-containing DNA, hexameric T antigen was inactive in these reactions
Antigen concentration was determined by scanning densitometryof silver-stained sodium dodecyl sulfate-polyacrylamide gels after elecplacement of a5'-32P-labeledoligonucleotide annealedto trophoresis of gradient fractions.DNA replication was measured by 4 x 1 7 4 single-stranded circular DNA) activities were meas- the formation of acid-insoluble[3H]dTMP.ATPaseactivity was ured across the glycerol gradient fractions, they corresponded measured by determiningthe formation of [cx-~'P]ADfProm [(U-~'P]
Summary
T antigen concentration was determined by scanning densitometryof silver-stained sodium dodecyl sulfate-polyacrylamide gels after elecplacement of a5'-32P-labeledoligonucleotide annealedto trophoresis of gradient fractions.DNA replication was measured by 4 x 1 7 4 single-stranded circular DNA) activities were meas- the formation of acid-insoluble[3H]dTMP.ATPaseactivity was ured across the glycerol gradient fractions, they corresponded measured by determiningthe formation of [cx-~'P]ADfProm [(U-~'P]-. When DNA replication activity was measured across the antigen hexamer (Fig. 4C). Replication activity was not observed using any a t 37 "C in the presence of 7 mM MgC12, but in the absence of the fractions of the third gradient,which contained the T of ATP (Fig. 4C). Replication activity could be restored t o a lesser degree by incubation of the fractions for 15, 30, or 45 min (data not shown)
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