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PMID: 19684079 Published · ppublish English Journal Article Research Support, N.I.H., Extramural Research Support, N.I.H., Intramural

A real-time fluorescence method for enzymatic characterization of specialized human DNA polymerases.

Nucleic acids research ·Vol. 37 ·No. 19 ·2009-10-00 ·Pages e128

Dorjsuren D, Wilson DM, Beard WA, McDonald JP, Austin CP, Woodgate R, Wilson SH, Simeonov A

Abstract

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 beta, eta, iota and kappa 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 iota and eta. 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.

MeSH Terms
Cations, Divalent DNA Damage DNA-Directed DNA Polymerase/metabolism Fluorometry/methods Humans Metals/chemistry
Chemicals
Cations, Divalent Metals DNA-Directed DNA Polymerase
Authors & Affiliations
8 authors, click to expand affiliations / ORCID
Dorjsuren Dorjbal
NIH Chemical Genomics Center, National Human Genome Research Institute, National Institutes of Health, Bethesda, MD 20892-3370, USA.
Wilson David M
Beard William A
McDonald John P
Austin Christopher P
Woodgate Roger
Wilson Samuel H
Simeonov Anton
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Article Info
Journal
Nucleic acids research
Abbr.
Nucleic Acids Res
ISSN
1362-4962
DOI
10.1093/nar/gkp641
Published
2009-10-00
Epub
2009-00-14
Pages
e128
Language
English
Region
England
NLM ID
0411011
PMCID
PMC2770649
Subset
IM
Grants
NCI NIH HHS · 1U19CA105010 · United States
NIEHS NIH HHS · Z01-ES050158 · United States
NIEHS NIH HHS · Z01-ES050161 · United States
Intramural NIH HHS · United States
External Links
PubMed source DOI: 10.1093/nar/gkp641 PMC Full Text View journal details
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