Nucleic Acids Research, 1991, Vol. 19, No. 12 3319-3323
© 1991
CHEMISTRY |
Stability of DNA thymine hydrates
Department of Pathology and Fels Institute for Cancer Research and Molecular Biology, Temple University School of Medicine Philadelphia, PA 19140, USA
* To whom correspondence should be addressed
Received March 11, 1991. Revised May 14, 1991. Accepted May 14, 1991.
Pyrimldine hydrates are products of ultraviolet irradiation of DNA. We have already demonstrated the formation of both cis-thymine hydrate and trans-thymine hydrate (6-hydroxy-5,6-dlhydrothymine) in Irradiated poly(dA-dT): poly(dA-dT). These are released from DNA as free bases by bacterial or human glycosylases. Thymine hydrate stabilities were studied in Irradiated DNA substrates using purified E. coll endonuclease III as a reagent for their removal. After irradiation, substrate poly(dA-dT): poly(dA-dT), radiolabeled In thymine, was incubated at 50, 60, 70 or 80°C, cooled, and then reacted with the enzyme under standard conditions. Thymine hydrates were assayed by enzymic release of labeled material into the ethanol-soluble fraction. Their identities were confirmed by high performance liquid chromatography. The decay of thymine hydrates in heated DNA followed first-order kinetics with a k = 2.8 x 10-5/sec at 80°C. These hydrates were also detected in lesser quantities In the unirradiated, control substrate. Extrapolation from an Arrhenlus plot yields an estimated half-life of 33.3 hours at 37°C for DNA thymine hydrates. Such stability, together with their formation in unirradiated DNA, suggest thymine hydrates to be formed under physiological conditions and to be sufficiently stable in DNA to be potentially genotoxic. This necessitates their constant removal from DNA by the excision-repair system.