Nucleic Acids Research, 1988, Vol. 16, No. 8 3327-3340
© 1988
Articles |
UV-induced photoproducts of 5-methylcytosine in a DNA sequence context
Department of Pathology, Section of Experimental Pathology, The University of Texas M.D.Anderson Hospital and Tumor Institute at Houston 1515 Holcombe Boulevard, Houston, TX 77030, USA
*To whom correspondence should be addressed
Received December 10, 1987. Revised March 16, 1988. Accepted March 16, 1988.
In order to detect possible m5C photoproducts, highly purified rat liver DNA-cytos1ne methyltransferase was used to specifically generate m5C with a radioactive methyl group. When these DNAs were subjected to a large dose (10 kJ/m2) of 254 nm or 302 nm ultraviolet light (UVB) to enhance the yield, two labeled photoproducts were detected and isolated by reverse phase HPLC after formic add hydrolysis. Further studies using acetone as a triplet state sensitizer and UVB irradiation suggested that photoproduct II was activated via a triplet state while the more polar photoproduct I was not. Photoreversion of the purified photoproducts with 10 kJ/m2 254 nm light demonstrated the following reactions: Photoproduct I regenerated m5C, while photoproduct II is split and regenerated nrC and photoproduct I. These results suggest that photoproduct I is monomeric while photoproduct II dimeric, and from the latter's elution position possibly a cyclobutyl type dimer arising from a reaction with an adjacent cytosine. Using d[TTG] and d[Cm5CG] as models of typical sequences, irradiation with 10 kJ/m2 254 nm or 302 nm, respectively, gave rise to a small component having altered mobility in sequencing gels. The altered mobility trinucleotides were resistant to degradation by PI and micrococcal nucleases as expected from photodimerization of the pyrimidine bases. Furthermore, oligonucleotide substrates containing m5C were synthesized and shown to be susceptible to T4 endonuclease v action at locations consistent with d[Cm5C] photodimer formation when irradiated in the UVB range.
+Present address: Department of Pharmacology, Mahidol University, Bangkok, Thailand