Liquid Chromatography‐Mass Spectrometry Analysis of DNA Polymerase Reaction Products
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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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Basic Protocol 1:
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Figure 7.16.1 Schematic representation of the approach used for the liquid chromatography‐mass spectrometry analysis of DNA polymerase reaction products. The first step involves the extension of a primer containing one or more dU residues across a lesion containing template by a DNA polymerase in the presence of all four dNTPs. Following primer extension, the DNA is cleaved into small pieces in a two‐step process to facilitate LC‐MS analysis. dU in the primer is first removed using UDG. The abasic sites formed are then converted into cleavage sites by hot piperidine. The short pieces of DNA that resulted from primer extension are finally analyzed using LC‐MS and LC‐MS/MS to determine their nucleotide sequences and relative yields. View Image -
Figure 7.16.2 CID of DNA fragments resulting in the generation of w and a‐B ions (Ni et al., ). (A ) CID fragmentation of specific bonds in DNA resulting in a‐B and w ions. (B ) Figure showing that fragmentation generally can occur at any base of the DNA fragment forming wn and an ‐Bn ions that can be used to determine the sequence of the DNA fragment. View Image -
Figure 7.16.3 Analysis of the extension product across O 6 ‐benzyl (Bz) G DNA lesion by human DNA polymerase η (Choi, ). (A ) Total ion chromatogram (TIC) of the reaction mixture. (B ) ESI‐LC‐mass spectrum of the products eluting at 3.90‐4.2 min in TIC spectrum shown in Figure A. (C ) TIC of the ESI‐LC‐MS/MS analysis of the m/z 1099.6 species. (D ) CID mass spectrum of m/z 1099.6 species. (E ) TIC of the ESI‐LC‐MS/MS analysis of the m/z 1103.3 species. (F ) CID mass spectrum of the m/z 1103.3 species. View Image -
Figure 7.16.4 Products of primer extension across O 6 ‐BzG DNA template by human DNA polymerase η and their relative yields (Choi et al., ). View Image
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Literature Cited
| Literature Cited | |
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| 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. | |
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| 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. | |
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| 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. | |
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| 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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