6-Thioguanine in DNA as CD-spectroscopic probe to study local structural changes upon protein binding.
A combination of experimental and theoretical circular dichroism (CD) spectroscopy was used to study local deformations of DNA caused by binding of the base flipping DNA methyltransferase M.TaqI. To selectively study the structural changes within the DNA, we replaced single guanine residues at six different positions in duplex DNA with 6-thioguanine (s(6)G), which absorbs at 342 nm where unmodified DNA and the enzyme are transparent. The shape and the transition wavelength of a CD signal around 340 nm in the spectra of the free DNA and the M.TaqI-bound DNA were found to depend on the position of the s(6)G probe. Theoretical rotational strengths were calculated employing the matrix method which is frequently used to model the CD of large biomolecules. The only chromophores in these calculations were the nucleic acid bases. Comparison of the measured and the calculated CD spectra showed that the applied computational method qualitatively reproduces the dominant band observed around 340 nm in all cases. From our results we conclude that the spectral changes observed upon binding of the enzyme to the DNA are indeed predominantly due to structural changes within the DNA and not to other effects caused by the presence of the enzyme.
Repges
Rudolf
R
Institute of Organic Chemistry, RWTH Aachen University, Landoltweg 1, D-52074 Aachen, Germany.
Beuck
Christine
C
Weinhold
Elmar
E
Raabe
Gerhard
G
Fleischhauer
Jorg
J
D002942:Circular Dichroism; D004247:DNA / Q000737:chemistry* / Q000378:metabolism*; D015252:Deoxyribonucleases, Type II Site-Specific / Q000737:chemistry* / Q000378:metabolism*; D008958:Models, Molecular; D009690:Nucleic Acid Conformation; D009696:Nucleic Acids / Q000737:chemistry; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D013824:Thermus / Q000201:enzymology; D013866:Thioguanine / Q000737:chemistry*
D009696:Nucleic Acids; D004247:DNA; D015252:Deoxyribonucleases, Type II Site-Specific; C027046:TCGA-specific type II deoxyribonucleases; D013866:Thioguanine