Nucleic Acids Research Advance Access originally published online on August 14, 2009
Nucleic Acids Research 2009 37(19):e128; doi:10.1093/nar/gkp641
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Nucleic Acids Research, 2009, Vol. 37, No. 19 e128
© Published by Oxford University Press 2009
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.
Methods Online |
A real-time fluorescence method for enzymatic characterization of specialized human DNA polymerases
1NIH Chemical Genomics Center, National Human Genome Research Institute, National Institutes of Health, Bethesda, MD 20892-3370, 2Laboratory of Molecular Gerontology, National Institute on Aging, National Institutes of Health, Baltimore, MD 21224, 3Laboratory of Structural Biology, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, NC 27709 and 4Laboratory of Genomic Integrity, Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD 20892-3371, USA
*To whom correspondence should be addressed. Tel: +1 301 217 5721; Fax: +1 301 217 5736; Email: asimeono{at}mail.nih.gov
Received May 13, 2009. Revised July 11, 2009. Accepted July 17, 2009.
Specialized DNA polymerases are involved in DNA synthesis during base-excision repair and translesion synthesis across a wide range of chemically modified DNA templates. Notable features of these enzymes include low catalytic efficiency, low processivity and low fidelity. Traditionally, in vitro studies of these enzymes have utilized radiolabeled substrates and gel electrophoretic separation of products. We have developed a simple homogeneous fluorescence-based method to study the enzymology of specialized DNA polymerases in real time. The method is based on fluorescent reporter strand displacement from a tripartite substrate containing a quencher-labeled template strand, an unlabeled primer and a fluorophore-labeled reporter. With this method, we could follow the activity of human DNA polymerases β, 
, 
and 
under different reaction conditions, and we investigated incorporation of the aberrant nucleotide, 8-oxodGTP, as well as bypass of an abasic site or 8-oxoG DNA template lesion in different configurations. Lastly, we demonstrate that the method can be used for small molecule inhibitor discovery and characterization in highly miniaturized settings, and we report the first nanomolar inhibitors of Y-family DNA polymerases and . The fluorogenic method presented here should facilitate mechanistic and inhibitor investigations of these polymerases and is also applicable to the study of highly processive replicative polymerases.