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.

National Institute of Health (NIH)
National Cancer Institute (NCI)
Research Program Projects (P01)
Project #
Application #
Study Section
Project Start
Project End
Budget Start
Budget End
Support Year
Fiscal Year
Total Cost
Indirect Cost
Sanders, M H; Bates, S E; Wilbur, B S et al. (2004) Repair rates of R-band, G-band and C-band DNA in murine and human cultured cells. Cytogenet Genome Res 104:35-45
Dai, S M; O'Connor, T R; Holmquist, G P et al. (2002) Ligation-mediated PCR: robotic liquid handling for DNA damage and repair. Biotechniques 33:1090-7
Komura , J; Ikehata, H; Hosoi, Y et al. (2001) Mapping psoralen cross-links at the nucleotide level in mammalian cells: suppression of cross-linking at transcription factor- or nucleosome-binding sites. Biochemistry 40:4096-105
Cloutier, J F; Castonguay, A; O'Connor, T R et al. (2001) Alkylating agent and chromatin structure determine sequence context-dependent formation of alkylpurines. J Mol Biol 306:169-88
Chen, J Z; Smith, L; Pfeifer, G P et al. (2001) Fluorescence-based directed termination PCR: direct mutation characterization without sequencing. Nucleic Acids Res 29:E17
Miao, F; Bouziane, M; Dammann, R et al. (2000) 3-Methyladenine-DNA glycosylase (MPG protein) interacts with human RAD23 proteins. J Biol Chem 275:28433-8
Bouziane, M; Miao, F; Bates, S E et al. (2000) Promoter structure and cell cycle dependent expression of the human methylpurine-DNA glycosylase gene. Mutat Res 461:15-29
Pfeifer, G P (2000) p53 mutational spectra and the role of methylated CpG sequences. Mutat Res 450:155-66
Szabo, P E; Pfeifer, G P; Miao, F et al. (2000) Improved in vivo dimethyl sulfate footprinting using AlkA protein: DNA-protein interactions at the mouse H19 gene promoter in primary embryo fibroblasts. Anal Biochem 283:112-6
Chen, J Z; Qiu, J; Shen, B et al. (2000) Mutational spectrum analysis of RNase H(35) deficient Saccharomyces cerevisiae using fluorescence-based directed termination PCR. Nucleic Acids Res 28:3649-56

Showing the most recent 10 out of 33 publications