Genomic DNA in mammalian cells is susceptible to damage from exposure to a variety of environmental agents and from byproducts of endogenous metabolism.  In this presentation, I will discuss our application of mass spectrometry, along with molecular biology and genetic tools, to assess the formation, repair and biological consequences of bulky DNA lesions induced by reactive oxygen species.  First, we employ LC-MSn with the stable isotope dilution technique for quantifying accurately the levels of these DNA lesions formed in animal tissues and cultured human cells.  Together with genetic manipulation using knockout mouse models or siRNA technology, the LC-MSn quantification method facilitates the delineation of pathways involved in the repair of the oxidatively induced bulky DNA lesions.  Second, we use shuttle vector technology, in conjunction with LC-MS/MS for product identification, to examine quantitatively how structurely defined DNA lesions alter the efficiency and fidelity of DNA replication and transcription in cells. By performing these experiments in a background deficient in certain repair factor(s) or translesion synthesis (TLS) polymerase(s), the method can also lead to the determination of the roles of a particular repair factor and TLS polymerase involved in the repair and replicative bypass of these DNA lesions in cells. The integrated chemical biology approach led to improved understanding about the biological implications of the oxidatively induced bulky DNA lesions.

Chemistry and biology of endogenous oxidatively induced DNA lesions

244th National Meeting (2012)

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