Resonance Raman spectroscopy of 4-thiothymidine and oligodeoxynucleotides containing this base both free in solution and bound to the restriction endonuclease EcoRV.
The resonance Raman spectra of 4-thiothymidine [4ST] have been recorded (a) in the free deoxynucleoside form, (b) when incorporated into the single stranded oligodeoxynucleotide d(AG[4ST]-TC), and (c) within the double-stranded self-complementary dodecamer d(GACGA[4ST]ATCGTC). Vibrational mode assignments of almost all the major Raman bands observed in each spectra have been made, mainly by comparison with the published assignments of related nucleosides and nucleotides. Differences between the spectra were observed, particularly when [4ST] and d(AG[4ST]TC) were compared to d(GACGA[4ST]ATCGTC). This is explained in terms of the variations in structure between single-and double-stranded DNA. Good quality spectra were obtained at nucleotide/oligonucleotide concentrations of between 100 and 500 microM and this coupled with an apparatus that uses small volumes (100 microL) allowed measurement of the spectrum of d(GACGA[4ST]ATCGTC) bound to the EcoRV endonuclease. This well characterised nuclease, for which crystal structures are available, recognizes d(GATAT) sequences. When this is replaced with d(GA[4ST]ATC), a poor substrate results but turnover can be prevented during data accumulation by omission of the essential cation Mg2+. Large shifts in several of the Raman bands were observed, and these have been related to the environment of the [4ST] base in the protein-bound oligonucleotide as deduced from the crystal structure. The wavenumber for the C = S stretch vibration in free d(GACGA[4ST]ATCGTC) has been used to calculate the strength of the Watson-Crick hydrogen bond between the sulphur atom in [4ST] and the 6-NH2 group on its partner dA. On binding to the enzyme, the shift in the wavenumber of the C = S stretch indicates this Watson-Crick hydrogen bond is weakened, in good agreement with X-ray structures. The advantage of using [4ST] as a resonance Raman probe is that it absorbs at 340 nm, a wavelength where other nucleic acid and protein absorbance is minimal. Thus the spectra obtained are very simple and consist of signals that arise predominantly from the thiobase alone, and this facilitates data interpretation.
Thorogood
H
H
Department of Biochemistry and Genetics, University of Newcastle, Newcastle upon Tyne, U.K.
Waters
T R
TR
Parker
A W
AW
Wharton
C W
CW
Connolly
B A
BA
D001483:Base Sequence; D015252:Deoxyribonucleases, Type II Site-Specific / Q000378:metabolism*; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides / Q000737:chemistry*; D011485:Protein Binding; D013059:Spectrum Analysis, Raman; D013816:Thermodynamics; D013872:Thionucleosides / Q000737:chemistry* / Q000378:metabolism; D013936:Thymidine / Q000031:analogs & derivatives* / Q000737:chemistry / Q000378:metabolism
D009838:Oligodeoxyribonucleotides; D013872:Thionucleosides; C070456:4'-thiothymidine; D015252:Deoxyribonucleases, Type II Site-Specific; C027440:GATATC-specific type II deoxyribonucleases; D013936:Thymidine