Nitric oxide-induced interstrand cross-links in DNA.
The DNA damaging effects of nitrous acid have been extensively studied, and the formation of interstrand cross-links have been observed. The potential for this cross-linking to occur through a common nitrosating intermediate derived from nitric oxide is investigated here. Using a HPLC laser-induced fluorescence (LIF) system, the amount of interstrand cross-link formed on nitric oxide treatment of the 5'-fluorescein-labeled oligomer ATATCGATCGATAT was determined. This self-complimentary sequence contains two 5'-CG sequences, which is the preferred site for nitrous acid-induced cross-linking. Nitric oxide was delivered to an 0.5 mM oligomer solution at 15 nmol/mL/min to give a final nitrite concentration of 652 microM. The resulting concentration of the deamination product, xanthine, in this sample was found to be 211 +/- 39 nM, using GC/MS, and the amount of interstrand cross-link was determined to be 13 +/- 2.5 nM. Therefore, upon nitric oxide treatment, the cross-link is found at approximately 6% of the amount of the deamination product. Using this system, detection of the cross-link is also possible for significantly lower doses of nitric oxide, as demonstrated by treatment of the same oligomer with NO at a rate of 18 nmol/mL/min resulting in a final nitrite concentration of 126 microM. The concentration of interstrand cross-link was determined to be 3.6 +/- 0.1 nM in this sample. Therefore, using the same dose rate, when the total nitric oxide concentration delivered drops by a factor of approximately 5, the concentration of cross-link drops by a factor of about 4-indicating a qausi-linear response. It may now be possible to predict the number of cross-links in a small genome based on the number of CpG sequences and the yield of xanthine derived from nitrosative deamination.
Caulfield
Jennifer L
JL
Biological Engineering Division, Massachusetts Institute of Technology, Cambridge 02139, USA.
Wishnok
John S
JS
Tannenbaum
Steven R
SR
D002851:Chromatography, High Pressure Liquid; D003432:Cross-Linking Reagents / Q000737:chemistry*; D004247:DNA / Q000737:chemistry*; D005452:Fluoresceins; D005453:Fluorescence; D005456:Fluorescent Dyes; D007834:Lasers; D009569:Nitric Oxide / Q000737:chemistry*; D009573:Nitrites / Q000032:analysis; D019820:Xanthine / Q000032:analysis
D003432:Cross-Linking Reagents; D005452:Fluoresceins; D005456:Fluorescent Dyes; D009573:Nitrites; D019820:Xanthine; D009569:Nitric Oxide; D004247:DNA