Published online 19 July 2004
Nucleic Acids Research, Vol. 32 No. 13 © Oxford University Press 2004; all rights reserved
Protein–DNA footprinting by endcapped duplex oligodeoxyribonucleotides
1 Department of Medicinal Chemistry and Molecular Pharmacology, Purdue University, West Lafayette, IN 47907, USA and 2 Walther Cancer Institute, Indianapolis, IN 46208, USA
* To whom correspondence should be addressed at Department of Medicinal Chemistry and Molecular Pharmacology, Purdue University, West Lafayette, IN 47907, USA. Tel: +1 765 494 6275; Fax: +1 765 494 9193; Email: bergstrom{at}purdue.edu
Received April 21, 2004; Revised and Accepted June 25, 2004
Oligodeoxyribonucleotides (5'-phosphorylated) of varying lengths were capped using a polyamide linker to form thermodynamically stable, endcapped DNA duplexes containing 8–14 bp. We have employed these endcapped DNA duplexes as tools to determine the DNA footprint of T4 DNA ligase. By high-performance liquid chromatography and PAGE analysis of the ligation mixtures of the endcapped DNA duplexes, we have found that by varying the lengths and the position of the nick, we can determine the minimal DNA-binding site as well as the mode of binding (symmetrical or asymmetrical binding) by the enzyme. The results of the study revealed that a 11 bp endcapped duplex was the shortest duplex effectively ligated. Dependence of ligation efficiency on nick position demonstrates that T4 DNA ligase bound asymmetrically to its DNA substrate. The use of a set of thermodynamically stable endcapped deoxyribonucleoside duplexes as a tool to elucidate the DNA footprint provides an efficient strategy for footprinting, which avoids ambiguities associated with chemical and biochemical footprinting methods.