Excision of ultraviolet-induced photoproducts of 5-methylcytosine from DNA.
Transition mutations at DNA 5-methylcytosines, congregated at CpG islands, are implicated in the etiogenesis of human diseases. Formation of 5-methylcytosine hydrate (5-methyl-6-hydroxy-5,6-dihydrocytosine) by hydration of the 5,6 double bond of 5-methylcytosine has been suggested as an intermediate in a possible mechanism of deamination to thymine. Ultraviolet irradiation of DNA yields pyrimidine hydrates, which are removed by repair glycosylases. We have identified 5-methylcytosine photoproducts following their excision from DNA by E. coli endonuclease III. Poly(dG-[3H]5-medC):poly(dG-[3H]5-medC) was irradiated and reacted with the enzyme. Radiolabeled photoproduct releases were directly proportional to irradiation doses and enzyme concentrations. These were identified as cis-thymine hydrate (6-hydroxy-5,6-dihydrothymine) and trans-thymine hydrate. Recovery of thymine hydrates is consistent with hydration of pyrimidines. Subsequent heating (which converts thymine hydrates to thymines) and chemical sequencing of an irradiated, 3' end-labeled, synthetic DNA strand demonstrated the appearance of thymine at the 5-methylcytosine site. These results demonstrate a mechanism for deamination of DNA 5-methylcytosine via hydration of the 5,6 double bond, putatively yielding 5-methylcytosine hydrate; this deaminates to thymine hydrate, and loss of water yields thymine formation at the 5-methylcytosine site. Identification of these DNA 5-methylcytosine modified moieties indicates a possible molecular mechanism for the frequent transition mutations found at CpG loci.
Vairapandi
M
M
Department of Pathology and Laboratory Medicine, Temple University School of Medicine, Philadelphia, PA 19140.
Duker
N J
NJ
D044503:5-Methylcytosine; D001483:Base Sequence; D003596:Cytosine / Q000031:analogs & derivatives* / Q000378:metabolism; D004247:DNA / Q000378:metabolism* / Q000528:radiation effects; D004260:DNA Repair / Q000502:physiology*; D043224:Deoxyribonuclease (Pyrimidine Dimer); D015226:Dinucleoside Phosphates / Q000378:metabolism; D004706:Endodeoxyribonucleases; D006801:Humans; D008745:Methylation; D008969:Molecular Sequence Data; D011089:Polydeoxyribonucleotides / Q000138:chemical synthesis / Q000378:metabolism / Q000528:radiation effects; D013941:Thymine / Q000031:analogs & derivatives / Q000032:analysis / Q000096:biosynthesis; D014466:Ultraviolet Rays
D044503:5-Methylcytosine; D003596:Cytosine; D004247:DNA; D043224:Deoxyribonuclease (Pyrimidine Dimer); D015226:Dinucleoside Phosphates; D004706:Endodeoxyribonucleases; D011089:Polydeoxyribonucleotides; D013941:Thymine; C066110:6-hydroxy-5,6-dihydrothymine; C015772:CpG dinucleotide; C019530:poly(dC-dG)