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The Development of a novel plasmid construction technique for analyzing repair of clustered DNA damage in vivo

Takahashi, Momoko; Shikazono, Naoya

DNA is damaged by various factors and unrepaired DNA damage would cause alterations of the genome, and thus various biological effects. To avoid these effects, there are diverse mechanisms to repair DNA damage in cells. Clustered DNA damage, defined as two or more lesions in one to two helical turns of DNA, is induced by ionizing radiation. Clustered DNA damage is considered to be refractory to repair and therefore highly lethal and mutagenic. However, the mechanism on how clustered DNA damage causes mutagenesis or lethality is still largely unknown. We constructed lesion-containing plasmids using a novel approach in order to analyze repair of clustered DNA damage. The method introduced by Karata was modified to construct the plasmid. We found that the constructed plasmid DNA had a defined lesion. As DNA lesions could be placed at any position on either strand, the method allows us to construct cluster DNA damage in a plasmid, and would be highly useful in analyzing ${it in vivo}$ processing of clustered DNA damage. We will discuss about the analysis of repair of clustered DNA damage in ${it E. coli}$, as an example.

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