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Assembly of the replication initiation complex on SV40 origin DNA.

Daniel T Simmons1, Dahai Gai, Rebekah Parsons

  • 1Department of Biological Sciences, University of Delaware, Newark, DE 19716-2590, USA. dsimmons@udel.edu

Nucleic Acids Research
|February 13, 2004
PubMed
Summary

DNA polymerase alpha/primase (pol/prim) is crucial for stabilizing the simian virus 40 replication initiation complex. It acts as the first cellular protein to bind, followed by topoisomerase I (topo I) and replication protein A (RPA).

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Area of Science:

  • Molecular Biology
  • Virology
  • Biochemistry

Background:

  • The precise assembly of protein complexes at the simian virus 40 origin to initiate DNA replication remains unclear.
  • This complex involves viral T antigen and cellular replication protein A (RPA), DNA polymerase alpha/primase (pol/prim), and topoisomerase I (topo I).

Purpose of the Study:

  • To elucidate the sequential binding order of proteins involved in simian virus 40 DNA replication initiation.
  • To understand the roles of pol/prim, topo I, and RPA in forming the replication initiation complex.

Main Methods:

  • Binding assays utilizing biotinylated simian virus 40 origin DNA.
  • Investigating protein interactions and complex formation in the presence of varying protein concentrations.

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Main Results:

  • DNA polymerase alpha/primase (pol/prim) is essential for stabilizing the initiation complex against topoisomerase I (topo I) activity.
  • Pol/prim modulates topo I binding; topo I appears to recruit replication protein A (RPA).
  • RPA has minimal effect on the recruitment of other proteins, suggesting pol/prim is the initial cellular protein to bind T antigen.

Conclusions:

  • The likely binding order is T antigen, followed by pol/prim, then topo I and RPA (or a topo I-RPA complex).
  • Two molecules of topo I bind per T antigen double hexamer.
  • DNA itself plays a role in recruiting topo I to the origin.