Influence of cytosine methylation on ultraviolet-induced cyclobutane pyrimidine dimer formation in genomic DNA.
The ultraviolet (UV) component of sunlight is the main cause of skin cancer. More than 50% of all non-melanoma skin cancers and >90% of squamous cell carcinomas in the US carry a sunlight-induced mutation in the p53 tumor suppressor gene. These mutations have a strong tendency to occur at methylated cytosines. Ligation-mediated PCR (LMPCR) was used to compare at nucleotide resolution DNA photoproduct formation at dipyrimidine sites either containing or lacking a methylated cytosine. For this purpose, we exploited the fact that the X chromosome is methylated in females only on the inactive X chromosome, and that the FMR1 (fragile-X mental retardation 1) gene is methylated only in fragile-X syndrome male patients. Purified genomic DNA was irradiated with UVC (254nm), UVB (290-320nm) or monochromatic UVB (302 and 313nm) to determine the effect of different wavelengths on cyclobutane pyrimidine dimer (CPD) formation along the X-linked PGK1 (phosphoglycerate kinase 1) and FMR1 genes. We show that constitutive methylation of cytosine increases the frequency of UVB-induced CPD formation by 1.7-fold, confirming that methylation per se is influencing the probability of damage formation. This was true for both UVB sources used, either broadband or monochromatic, but not for UVC. Our data prove unequivocally that following UVB exposure methylated cytosines are significantly more susceptible to CPD formation compared with unmethylated cytosines.
Division of Pathology, Department of Medical Biology, Université Laval, Québec, QC, Canada.
Patrick J
PJ
Rochette
Sandrine
S
Lacoste
Jean-Philippe
JP
Therrien
Nathalie
N
Bastien
Douglas E
DE
Brash
Régen
R
Drouin
D001483:Base Sequence; D002478:Cells, Cultured; D041321:Chromosomes, Human, X / Q000378:metabolism / Q000528:radiation effects; D003596:Cytosine / Q000378:metabolism*; D004249:DNA Damage; D019175:DNA Methylation / Q000528:radiation effects*; D017931:DNA Primers / Q000235:genetics; D005260:Female; D051860:Fragile X Messenger Ribonucleoprotein 1 / Q000235:genetics; D005600:Fragile X Syndrome / Q000235:genetics / Q000378:metabolism; D006801:Humans; D008297:Male; D010735:Phosphoglycerate Kinase / Q000235:genetics; D011740:Pyrimidine Dimers / Q000378:metabolism* / Q000528:radiation effects*; D014466:Ultraviolet Rays / Q000009:adverse effects*; D049951:X Chromosome Inactivation / Q000235:genetics
D003596:Cytosine; D017931:DNA Primers; D051860:Fragile X Messenger Ribonucleoprotein 1; D010735:Phosphoglycerate Kinase; D011740:Pyrimidine Dimers; C497683:FMR1 protein, human