Correlating and measuring DNA damage and mutations




Kotturi, Gopaul

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Mutations are generally thought to be targeted events. The distribution of mutations is based upon the initial original deposition of DNA damage and the fidelity and efficiency of repair of this damage. These factors are dependent on the primary site of DNA modification and the surrounding nucleotides (i.e., mutation is “context sensitive"). To better understand mutagenesis, I measured DNA damage and/or mutation at the DNA sequence level, then considered the impact of mutation location and the surrounding nucleotide environment. The selected mutagens, ultraviolet light (UV) and benzo(a)pyrene [B(a)P], were chosen because they produce well-characterized lesions and are environmentally relevant. UVC (254nm) light-induced DNA damage is well documented. UVC produces a characteristic spectrum of mutations. The predominant UV-induced mutations are C → T transitions occurring at TC or CC sites, as well as CC → TT tandem transitions. The latter class of mutation is considered the hallmark of UV mutagenesis. Quantitatively speaking, the primary types of UV-induced DNA lesions are cyclobutane pyrimidine dimers (CPDs) and the 6-4 pyrimidine/pyrimidone (64PyPy). These are also the suspected predominant pre-mutagenic lesions. Each lesion was independently measured at the DNA sequence level in a defined region of DNA. The pattern of UVC-induced DNA damage revealed a complex induction pattern. The flanking DNA nucleotides partially influenced the pattern of damage deposition. Sites where C → T transitions and CC → TT transitions were recovered at high frequencies were also frequently damaged. Thus, at these sites, mutation fixation was potentially more influenced by initial DNA damage than the rate of DNA repair. Two other components of the UV spectrum [UVB (290-320 nm) and UVA (320-400 nm)] are more environmentally relevant than UVC since UVB and UVA reach the surface of the earth.The results of UVC experiments were used as a guide to interpret the results obtained using UVB since direct light absorption by DNA has been shown to be one of the main biological effect at both wavelengths. The model that was chosen for the studies was an in vivo transgenic rodent mutagenesis assay. The research presented in the thesis represents one of the first studies to characterize UV-induced mutation at the DNA sequence level in rodent skin. The backs of female C57B1/6 lacl transgenic mice were shaved and exposed to either UVB or UVA light. UVB was found to be significantly more mutagenic than UVA. The UVB-induced mutation spectrum was characterized by C → T transitions at dipyrimidine sites, implicating CPD and/or 64PyPy lesions as premutational DNA lesions. The majority of UVA-induced mutations was C → T transitions at dipyrimidines sites and hence, as with UVB-induced mutation, attributed to CPDs and 64PyPy. In the UVA-dose response experiments, the induced mutant frequency was lower than expected at higher doses. A statistically significant increase in putative clonal expansion suggested that skin cells might have undergone cell killing followed by repopulation. In a final study, C57Bl/6 lacI transgenic male mice were intraperitoneally injected with the mutagen B(a)P at doses of 0, 62.5, 125, 250, and 500 mg/kg. This resulted in a linear increase in mutation frequency (4.8 to 53 × 10⁻⁵). All mutations increased at GC basepairs and not AT basepairs following B(a)P treatment. This was consistent with models suggesting guanosine adducts to be mutagenic lesions. In conclusion, the transgenic lacI mouse mutagenesis model was a sensitive target for in vivo mutagenesis from UVB, UVA and benzo(a)pyrene exposures. The system detected class-specific mutation frequency differences and increases in cell proliferation after mutagen exposure. With a further refinement of techniques, the correlation of DNA damage and mutation will allow even more exquisite studies.



DNA damage, Mutation (Biology)