MECHANISMS OF ULTRAVIOLET LIGHT INDUCED P53 MUTATIONS

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We developed ligation-mediated PCR (LMPCR) to map the frequency of mutagen-induced damage at nucleotide resolution and showed that the repair rate of UV-induced cyclopyrimidine dimers varies over fifteen fold from nucleotide position to nucleotide position. UV-induced cyclobutane dimers, if not repaired and subsequently misread by DNA polymerase result in mutations which, if they confer a tumorigenic growth advantage, appear in non-melanoma skin tumors. The major component of the UV-induced mutational signature is C->T transitions at dipyrimidine sites. We show that cyclobutane dimer repair rates along the p53 gene are highly correlated with the frequency each position appears as a C->T transition in the p53 mutation data base for non-melanoma skin tumors, but not for internal tumors. Thus, variation in cyclobutane dimer repair rate along the p53 gene drives a very significant fraction of the variation of the nucleotide position to nucleotide position mutation rate, mutational spectrum, of p53 mutations in skin tumors. With chronic UV doses, each nucleotide position reaches a steady state lesion equilibrium frequency, Leq. On finding that the kinetics of repair at any one nucleotide position are first order kinetics, we were able to show mathematically that Leq is the product of ease of damage after an acute UV dose times the half life for repair. As such, L incorporates two of the four sequential steps of mutagenesis and should be more highly correlated with C->T transition frequency than is repair rate alone. We will use ligation-mediated PCR to map Leq, Lacute, and repair rate in UV-B irradiated fibroblasts and keratinocytes using ligation-mediated PCR and determine which of these mutagenesis metrics best predicts the C->T transition frequency in the p53 mutation data base for non-melanoma skin tumors. Since cyclopyrimidine dimer repair rates vary with chronic dose strength (the adaptive response) and the environmentally relevant dose is a low one, we will map Leq at low UV doses to determine the correlation coefficient of the low dose Leq. To measure this, we will increase the sensitivity of LMPCR by size purification of p53 containing restriction fragments.