In Vitro Analysis of SV40 DNA Replication

Yi‐Ling Yin1, Anindya Dutta2

1 Genetics Institute, Cambridge, Massachusetts, 2 Brigham and Women's Hospital, Boston, Massachusetts
Publication Name:  Current Protocols in Cell Biology
Unit Number:  Unit 11.5
DOI:  10.1002/0471143030.cb1105s02
Online Posting Date:  May, 2001
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Abstract

This unit describes a method for studying cellular factors that affect eukaryotic replication by using a plasmid that contains an SV40 origin of replication and supplying the viral helicase T antigen. Replication factors are supplied by S100 extracts of 293 cells. The unit also contains protocols for expression and purification of T antigen and analysis of the products of replication.

     
 
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Table of Contents

  • Basic Protocol 1: In Vitro Replication of Plasmids Bearing SV40 Origin Sequences
  • Alternate Protocol 1: Analysis of DNA Replication Reaction Products by Alkaline Denaturing Agarose Gel
  • Support Protocol 1: Preparing 293 Cell Suspension Culture
  • Support Protocol 2: Preparation of a Replication‐Competent S100 Cell Extract
  • Support Protocol 3: Expression of Recombinant T Antigen
  • Support Protocol 4: Immunoaffinity Purification of T Antigen
  • Support Protocol 5: Preparation of Pab419 Immunoaffinity Column
  • Reagents and Solutions
  • Commentary
  • Literature Cited
     
 
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Materials

Basic Protocol 1: In Vitro Replication of Plasmids Bearing SV40 Origin Sequences

  Materials
  • 5× replication buffer (see recipe)
  • Supercoiled SV40 origin–containing DNA template
  • 2 U/µl creatine phosphokinase in 10 mM Tris⋅Cl, pH 8.0/50% (w/v) glycerol
  • Recombinant T antigen (see protocol 6)
  • 32P]dATP (800 or 6000 Ci/mmol; final volume 0.0125 ml)
  • Replication‐competent S100 cell extract (see protocol 4)
  • DE81 paper (Whatman)
  • 0.5 M Na 2HPO 4
  • 100% ethanol
  • Scintillation cocktail

Alternate Protocol 1: Analysis of DNA Replication Reaction Products by Alkaline Denaturing Agarose Gel

  • 3× stop solution (see recipe)
  • 20 mg/ml high‐quality glycogen (Life Technologies)
  • 5 M NaCl ( appendix 2A)
  • TE buffer, pH 8.0 ( appendix 2A)
  • Agarose
  • 1 N NaOH
  • 0.5 M EDTA ( appendix 2A)
  • 2× alkaline loading buffer (see recipe)
  • Alkaline running buffer, made fresh (see recipe)
  • 7% trichloracetic acid (TCA)
  • Additional reagents and equipment for phenol/chloroform extraction and ethanol precipitation of DNA ( appendix 3A), agarose gel electrophoresis ( appendix 3A), and autoradiography (unit 6.3)

Support Protocol 1: Preparing 293 Cell Suspension Culture

  Materials
  • Ten 10‐cm plates of confluent 293 cell culture
  • DMEM with 5% (v/v) calf serum
  • Joklik's medium (JRH Bioscience) with 5% (v/v) calf serum
  • Additional reagents and equipment for trypsinizing monolayer cells (unit 1.1), counting cells, and assessing viability (unit 1.1).

Support Protocol 2: Preparation of a Replication‐Competent S100 Cell Extract

  Materials
  • 8 liters mid‐log‐phase 293 cells (see protocol 3)
  • PBS ( appendix 2A), 4°C
  • Hypotonic lysis buffer (see recipe), 4°C
  • 5 M NaCl
  • Protein assay dye reagents (Bio‐Rad)
  • IEC PR7000 and rotor or equivalent
  • IEC Clinical centrifuge or equivalent
  • 40‐ml Dounce homogenizer (type B pestle)
  • RC5B Sorvall centrifuge with SS34 rotor
  • Beckman ultracentrifuge with SW 55.1 rotor

Support Protocol 3: Expression of Recombinant T Antigen

  Materials
  • Insect cells (Hi‐five cells, Invitrogen)
  • Grace's medium with 10% fetal bovine serum (FBS)
  • Recombinant T antigen baculovirus stock, high titer
  • Serum‐free medium

Support Protocol 4: Immunoaffinity Purification of T Antigen

  Materials
  • 1 ml Sepharose CL‐4B (Amersham Pharmacia Biotech) column packed in a 3‐ml syringe plugged with siliconized glass wool
  • Pab419–protein A–Sepharose (Pab419‐PAS) immunoaffinity column (see protocol 7)
  • T antigen lysis buffer (see recipe), 4°C
  • Insect cell pellet containing recombinant T antigen (see protocol 5)
  • Wash buffer 1 (see recipe), 4°C
  • Wash buffer 2 (see recipe), 4°C
  • Elution buffer (see recipe), 4°C
  • Protein assay dye reagent (Bio‐Rad)
  • Dialysis buffer (see recipe), 4°C
  • 0.1 M sodium borate, pH 9.0
  • 0.5 M piperazine‐N,N′‐bis(2‐hydroxypropanesulfonic acid) (PIPES), pH 7.0, 4°C
  • Dialysis tubing

Support Protocol 5: Preparation of Pab419 Immunoaffinity Column

  Materials
  • Pab419 ascites
  • Protein A–Sepharose (PAS; 1 ml packed volume) equilibrated in coupling buffer
  • Coupling buffer (see recipe)
  • recipeCoupling buffer with 0.5% (v/v) NP‐40 (4.2 ml of 10% NP‐40 in 100 ml buffer)
  • 0.1 M sodium borate, pH 9.0
  • 400 mM dimethylpimelimidate (freshly prepared)
  • 0.2 M ethanolamine, pH 8.0
  • 0.2% sodium azide
  • 5‐ml syringe plugged with silanized glass wool
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Figures

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Literature Cited

Literature Cited
   Borowiec, J.A. and Hurwitz, J. 1988. ATP stimulates the binding of simian virus 40 (SV40) large tumor antigen to the SV40 origin of replication. Proc. Natl. Acad. Sci. U.S.A. 85:,64‐68.
   Bullock, P.A., Seo, Y.S., and Hurwitz, J. 1991. Initiation of simian virus 40 DNA synthesis in vitro. Mol. Cell. Biol. 11:2350‐2356.
   Dean, F.B., Bullock, P., Murakami, Y., Wobbe, C.R., Weissbach, L., and Hurwitz, J. 1987. Simian virus 40 (SV40) DNA replication: SV40 large T antigen unwinds DNA containing the SV40 origin of replication. Proc. Natl. Acad. Sci. U.S.A. 84:16‐20.
   Dean, F.B. and Hurwitz, J. 1991. Simian virus 40 large T antigen untwists DNA at the origin of DNA replication. J. Biol. Chem. 266:5062‐5071.
   Denis, D. and Bullock, P.A. 1993. Primer‐DNA formation during simian virus 40 DNA replication in vitro. Mol. Cell. Biol. 13:2882‐2890.
   Dornreiter, I., Copeland, W.C., and Wang, T.S. 1993. Initiation of simian virus 40 DNA replication requires the interaction of a specific domain of human DNA polymerase alpha with large T antigen. Mol.Cell. Biol. 13:809‐820.
   Dutta, A. and Stillman, B. 1992. cdc2 family kinases phosphorylate a human cell DNA replication factor, RPA, and activate DNA replication. EMBO J. 11:189‐199.
   Goetz, G.S., Dean, F.B., Hurwitz, J., and Matson, S.W. 1998. The unwinding of duplex regions in DNA by the simian virus 40 large tumor antigen–associated DNA helicase activity. J. Biol. Chem. 263:383‐392.
   Krude, T., Jackman, M., Pines, J., and Laskey, R.A. 1997. Cyclin/Cdk‐dependent initiation of DNA replication in a human cell‐free system. Cell 88:109‐119.
   Lee, S.H., Kwong, A.D., Pan, Z.Q., and Hurwitz, J. 1991. Studies on the activator 1 protein complex, an accessory factor for proliferating cell nuclear antigen–dependent DNA polymerase delta. J. Biol.Chem. 266:594‐602.
   Li, J.J. and Kelly, T.J. 1984. Simian virus 40 DNA replication in vitro. Proc. Natl. Acad. Sci. U.S.A. 81:6973‐6977.
   Mossi, R., Jonsson, Z.O., Allen, B.L., Hardin, S.H., and Hubscher, U. 1997. Replication factor C interacts with the C‐terminal side of proliferating cell nuclear antigen. J. Biol. Chem. 272:1769‐1776.
   Murakami, Y., Eki, T., and Hurwitz, J. 1992. Studies on the initiation of simian virus 40 replication in vitro: RNA primer synthesis and its elongation. Proc. Natl. Acad. Sci. U.S.A. 89:952‐956.
   Tsurimoto, T. and Stillman, B. 1991a. Replication factors required for SV40 DNA replication in vitro. I. DNA structure–specific recognition of a primer‐template junction by eukaryotic DNA polymerases and their accessory proteins. J. Biol. Chem. 266:1950‐1960.
   Tsurimoto, T. and Stillman, B. 1991b. Replication factors required for SV40 DNA replication in vitro. II. Switching of DNA polymerase alpha and delta during initiation of leading and lagging strand synthesis. J. Biol. Chem. 266:1961‐1968.
   Turchi, J.J., Huang, L., Murante, R.S., Kim, Y., and Bambara, R.A. 1994. Enzymatic completion of mammalian lagging‐strand DNA replication. Proc. Natl. Acad. Sci. U.S.A. 91:9803‐9807.
   Wold, M.S., Li, J.J., and Kelly, T.J. 1987. Initiation of simian virus 40 DNA replication in vitro: large‐tumor‐antigen‐ and origin‐dependent unwinding of the template. Proc. Natl. Acad. Sci. U.S.A. 84:3643‐3647.
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