EDTA-derivatized deoxythymidine as a tool for rapid determination of protein binding polarity to DNA by intermolecular paramagnetic relaxation enhancement.
EDTA-derivatized deoxythymidine (dT-EDTA), incorporated into DNA and complexed to Fe2+ in the presence of dithiothreitol, is a widely used reagent for sequence-specific cleavage of duplex DNA. Using HPLC/electrospray mass spectrometry, we show that cleavage is specific to Fe2+, and no cleavage occurs when DNA-EDTA is complexed to other metal ions such as Ca2+, Mn2+, and Fe3+ even after many days. Because dT-EDTA can be incorporated at any desired position of a synthetic oligonucleotide, DNA-EDTA is ideally suited for the measurement of intermolecular paramagnetic relaxation enhancement effects between a paramagnetic ion chelated to DNA-EDTA and a bound protein. Measurements on the SRY/DNA-EDTA complex using two double-stranded oligonucleotides bearing dT-EDTA at opposite ends of the sequence indicate that intermolecular 1HN-T2 enhancement by chelated Mn2+ can be used to readily ascertain the polarity of protein binding to DNA and to derive quantitative long-range distance information for structure refinement. In the case of the SRY-DNA complex, excellent agreement between observed and calculated 1HN-T2 paramagnetic relaxation enhancement data can be achieved with insignificant shifts in the atomic coordinates ( approximately 0.25 A for all heavy atoms) while simultaneously satisfying all other experimental restraints. A unique feature of DNA-EDTA is that the relaxation enhancement effect can be tuned by judicious choice of the paramagnetic metal ion, thereby permitting a wide range of long-range intermolecular electron-proton distances, ranging from approximately 9 to 35 A, to be probed.
Iwahara
Junji
J
Laboratories of Chemical Physics and Bioorganic Chemistry, National Institute of Diabetes and Digestive and Kidney Diseases/NIH, Bethesda, MD 20892-0510, USA.
Anderson
D Eric
DE
Murphy
Elizabeth C
EC
Clore
G Marius
GM
D002118:Calcium / Q000737:chemistry; D002614:Chelating Agents / Q000737:chemistry; D004247:DNA / Q000737:chemistry / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000378:metabolism; D004492:Edetic Acid / Q000031:analogs & derivatives* / Q000737:chemistry; D008345:Manganese / Q000737:chemistry; D019906:Nuclear Magnetic Resonance, Biomolecular / Q000379:methods*; D009687:Nuclear Proteins; D009690:Nucleic Acid Conformation; D011485:Protein Binding; D051876:Sex-Determining Region Y Protein; D013936:Thymidine / Q000031:analogs & derivatives* / Q000737:chemistry; D014157:Transcription Factors
D002614:Chelating Agents; D004268:DNA-Binding Proteins; D009687:Nuclear Proteins; D051876:Sex-Determining Region Y Protein; D014157:Transcription Factors; D008345:Manganese; D004247:DNA; D004492:Edetic Acid; D002118:Calcium; D013936:Thymidine