Liquid Chromatography‐Mass Spectrometry Analysis of DNA Polymerase Reaction Products

Goutam Chowdhury1, F. Peter Guengerich1

1 Department of Biochemistry and Center in Molecular Toxicology, Vanderbilt University School of Medicine, Nashville, Tennessee
Publication Name:  Current Protocols in Nucleic Acid Chemistry
Unit Number:  Unit 7.16
DOI:  10.1002/0471142700.nc0716s47
Online Posting Date:  December, 2011
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Abstract

This unit describes experimental and analytical procedures for characterizing the efficiency and fidelity of translesion DNA synthesis across various DNA damages by DNA polymerases in vitro. This procedure utilizes primer extension assays followed by LC‐MS and LC‐MS/MS analysis of the extension products. Detailed explanations for the analysis of the LC‐MS/MS data for deciphering the nucleotide sequences of the DNA fragments are also presented. This approach provides a significant improvement over conventional methods, as it allows detection of misincorporation, as well as frameshift products. Curr. Protoc. Nucleic Acid Chem. 47:7.16.1‐7.16.11. © 2011 by John Wiley & Sons, Inc.

Keywords: DNA polymerase; translesion synthesis; sequencing; LC‐MS

     
 
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Table of Contents

  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1:

  Materials
  • DNA template (see Strategic Planning)
  • Primer (see Strategic Planning)
  • Tris⋅Cl buffer, pH 7.7 (see recipe)
  • DNA polymerase
  • Dithiothreitol (DTT)
  • NaCl
  • MgCl 2
  • Bovine serum albumin (BSA; Sigma)
  • HPLC‐grade water (Fisher Scientific)
  • 2′‐Deoxyoribonucleotide triphosphates (dNTP; Sigma)
  • Uracil‐DNA glycosylase (UDG; Sigma)
  • Piperidine (Sigma‐Aldrich)
  • Acetonitrile (CH 3CN), HPLC grade (Fisher Scientific)
  • Ammonium acetate (NH 4CH 3CO 2) (Fisher Scientific)
  • 2′‐Deoxyoligonucleotide (Midland)
  • Heating block
  • 37°C incubator
  • Bio‐Spin 6 spin column (Bio‐Rad)
  • Swinging‐bucket centrifuge
  • Centrifugal lyophilizer device [e.g., Centrivac (Labconco) or SpeedVac]
  • Acquity ultraperformance liquid chromatography (UPLC) system (Waters Associates)
  • Finnigan LTQ mass spectrometer (Thermo Fisher)
  • Acquity BEH octadecylsilane (C18) UPLC column (1.7 µm particle size, 1.0 mm × 100 mm; Waters Associates)
  • Oligo Composition Calculator v1.2 software (http://library.med.utah.edu/masspec/)
  • Mongo Oligo Mass Calculator v2.0 (http://library.med.utah.edu/masspec/mongo.htm)
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Figures

Videos

Literature Cited

Literature Cited
   Boosalis, M.S., Petruska, J., and Goodman, M.F. 1987. DNA polymerase insertion fidelity: Gel assay for site‐specific kinetics. J. Biol. Chem. 262:14689‐14696.
   Choi, J.‐Y., Chowdhury, G., Zang, H., Angel, K.C., Vu, C.C., Peterson, L.A., and Guengerich, F.P. 2006. Translesion synthesis across O6‐alkylguanine adducts by recombinant human DNA polymerases. J. Biol. Chem. 281:38244‐38256.
   Chowdhury, G. and Guengerich, F.P. 2008. Direct detection and mapping of sites of base modification in DNA fragments by tandem mass spectrometry. Angew. Chem. Int. Ed. 47:381‐384.
   Chowdhury, G. and Guengerich, F.P. 2009. Tandem mass spectrometry‐based detection of c4′‐oxidized abasic sites at specific positions in DNA fragments. Chem. Res. Toxicol. 22:1310‐1319.
   Christov, P.P., Angel, K.C., Guengerich, F.P., and Rizzo, C.J. 2009. Replication of the N5‐methyl‐formamidopyrimidine lesion of deoxyguanosine by DNA polymerases: An improved procedure for sequence analysis of in vitro bypass products by mass spectrometry. Chem. Res. Toxicol. 22:1086‐1095.
   Ellington, A. and Pollard, J.D. 1998. Purification of oligonucleotides using denaturing polyacrylamide gel electrophoresis. Curr. Protoc. Mol. Biol. 42:2.12.1‐2.12.7.
   Friedberg, E.C., Walker, G.C., Siede, W., Wood, R.D., Schultz, R.A., and Ellenberger, T. 1995. DNA Repair and Mutagenesis. American Society for Microbiology, Washington, D.C.
   Guengerich, F.P. 2006. Interactions of carcinogen‐bound DNA with individual DNA polymerases. Chem. Rev. 106:420‐452.
   Ni, J., Pomerantz, S.C., Rozenski, J., Zhang, Y., and Mc Closkey, J.A. 1996. Interpretation of oligonucleotide mass spectra for determination of sequence using electrospray ionization and tandem mass spectrometry. Anal. Chem. 68:1989‐1999.
   Searle, C.E. 1984. Chemical Carcinogens, Vol. 1 and 2. American Chemical Society, Washington, D.C.
   Zang, H., Goodenough, A.K., Choi, J.‐Y., Irminia, A., Loukachevitch, L.V., Kozekov, I.D., Angel, K.C., Rizzo, C.J., Egli, M., and Guengerich, F.P. 2005. DNA adduct bypass polymerization by Sulfolobus solfataricus DNA polymerase Dpo4. Analysis and crystal structures of multiple base‐pair substitution and frameshift product with the adduct 1,N2‐ethenoguanine. J. Biol. Chem. 280:29750‐29764.
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