Nucleic Acids Research Advance Access originally published online on April 22, 2007
Nucleic Acids Research 2007 35(9):3118-3127; doi:10.1093/nar/gkm168
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Nucleic Acids Research, 2007, Vol. 35, No. 9 3118-3127
© 2007 The Author(s)
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
Nucleic Acid Enzymes |
Nucleoside alpha-thiotriphosphates, polymerases and the exonuclease III analysis of oligonucleotides containing phosphorothioate linkages
Foundation for Applied Molecular Evolution, 1115 NW 4th Street, Gainesville, FL 32604-1174, USA
*To whom correspondence should be addressed at Foundation for Applied Molecular Evolution, P.O. Box 13174, Gainesville FL 32604-1174, USA Tel: +1 352 271 7005; Fax: +1 352 271 7076; Email: sbenner{at}ffame.org
Received January 15, 2007. Revised February 16, 2007. Accepted March 6, 2007.
The use of DNA polymerases to incorporate phosphorothioate linkages into DNA, and the use of exonuclease III to determine where those linkages have been incorporated, are re-examined in this work. The results presented here show that exonuclease III degrades single-stranded DNA as a substrate and digests through phosphorothioate linkages having one absolute stereochemistry, assigned (assuming inversion in the polymerase reaction) as S, but not the other absolute stereochemistry. This contrasts with a general view in the literature that exonuclease III favors double-stranded nucleic acid as a substrate and stops completely at phosphorothioate linkages. Furthermore, not all DNA polymerases appear to accept exclusively the (R) stereoisomer of nucleoside alpha-thiotriphosphates [and not the (S) diastereomer], a conclusion inferred two decades ago by examination of five Family-A polymerases and a reverse transcriptase. This suggests that caution is appropriate when extrapolating the detailed behavior of one polymerase from the behaviors of other polymerases. Furthermore, these results provide constraints on how exonuclease III–thiotriphosphate–polymerase combinations can be used to analyze the behavior of the components of a synthetic biology.