Rapid Isolation, Hydrolysis and Chromatography of Formaldehyde-Modified DNA

摘要

Deoxyadenosine, deoxyguanosine, deoxycytidine and thymidine were reacted with formaldehyde. High pressure liquid chromatographic (HPLC) analysis indicated that each deoxynucleoside had formed one major product. With the exception of the thymidine product, these adducts were analyzed by nuclear magnetic resonance spectroscopy and identified as hydroxymethyl derivatives at the exocyclic amines. Calf thymus DNA was incubated with (3H) formaldehyde and, after purification, enzymatically hydrolyzed to nucleosides. HPLC analysis indicated the presence of a substantial proportion of noncovalently-bound formaldehyde and the following hydroxymethyl adducts, listed in order of decreasing concentration: N6-hydroxymethyldeoxyadenosine >> N4-hydroxymethyldeoxycytidine > N2- hydroxymethyldeoxyguanosine. Incubation of Chinese hamster ovary (CHO) cells with (3H) formaldehyde resulted in metabolic incorporation of the formaldehyde into purines and pryimidines plus an appreciable concentration of formaldehyde noncovalently associated with the DNA. However, HPLC analysis clearly indicated the presence of N6-hydroxymethyldeoxyadenosine in the CHO cell genome.