Protein-DNA footprinting by endcapped duplex oligodeoxyribonucleotides.
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.
Ng
Pei-Sze
PS
Department of Medicinal Chemistry and Molecular Pharmacology, Purdue University, West Lafayette, IN 47907, USA.
Bergstrom
Donald E
DE
D001483:Base Sequence; D001665:Binding Sites; D002851:Chromatography, High Pressure Liquid; D004247:DNA / Q000737:chemistry / Q000378:metabolism; D018983:DNA Footprinting / Q000379:methods*; D011088:DNA Ligases / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism*; D009838:Oligodeoxyribonucleotides / Q000737:chemistry*; D011092:Polyethylene Glycols / Q000494:pharmacology; D011485:Protein Binding
D004268:DNA-Binding Proteins; D009838:Oligodeoxyribonucleotides; D011092:Polyethylene Glycols; D004247:DNA; D011088:DNA Ligases