SV40 DNA Replication In Vitro

  • Mark K. Kenny
  • Ann D. Kwong
  • Suk-Hee Lee
  • Takashi Matsumoto
  • Yeon Soo Seo
  • James A. Borowiec
  • Peter Bullock
  • Frank B. Dean
  • Toshihiko Eki
  • Yukio Ishimi
  • Jerard Hurwitz
Part of the NATO ASI Series book series (volume 34)

Abstract

Simian virus 40 (SV40) DNA replication has been reconstituted in vitro using purified proteins. SV40 large T antigen (TAg), DNA polymerase α-primase complex (pol α-primase), HeLa single-stranded DNA binding protein (HeLa SSB), and topo- isomerase I or II (topo I or II) produced extensive DNA synthesis when SV40 origin- containing DNA (ori+ DNA) was used as a template. Further addition of DNA ligase, RNase H, and a 5′→3′ exonuclease resulted in relaxed covalently closed circular DNA products (RFI’). Replication in vitro with purified proteins mimicked bona fide SV40 DNA replication in vivo by the following criteria: TAg dependency, functional SV40 origin dependency, aphidicolin sensitivity, leading and lagging strand semi-discontinuous synthesis, bidirectional replication from the origin, and covalently closed circular monomer products. Additionally, the species specificity of SV40 and polyoma virus DNA replication observed in vivo was also seen in vitro and can be attributed to the source of the pol α-primase.

The early steps leading to initiation of SV40 DNA replication have been investigated. Tag was found to form a double hexamer structure at the origin of replication in an ATP_dependent manner. The origin DNA contained within the nucleoprotein complex was found to be locally melted. Upon addition of an SSB and a topo which can relax positive supercoils, extensive unwinding of the DNA was observed.

Proliferating cell nuclear antigen (PCNA) was not required for replication with purified proteins; however, it was required for extensive DNA synthesis when crude fractions were used. PCNA was found to overcome an inhibitor present in crude fractions which blocks DNA chain elongation. The possible significance of this finding with regard to the regulation of DNA synthesis is discussed.

Keywords

Hydrolysis Sucrose Glycerol Agarose Electrophoresis 

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Copyright information

© Springer-Verlag Berlin Heidelberg 1989

Authors and Affiliations

  • Mark K. Kenny
    • 1
  • Ann D. Kwong
    • 1
  • Suk-Hee Lee
    • 1
  • Takashi Matsumoto
    • 1
  • Yeon Soo Seo
    • 1
  • James A. Borowiec
    • 1
  • Peter Bullock
    • 1
  • Frank B. Dean
    • 1
  • Toshihiko Eki
    • 1
  • Yukio Ishimi
    • 1
  • Jerard Hurwitz
    • 1
  1. 1.Graduate Program in Molecular BiologySloan-Kettering Cancer CenterNew YorkUSA

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