Construction of plasmid containing clustered DNA damage to analyze its in vivo repair

クラスターDNA損傷の生体内修復を調べるためのプラスミド作製法の開発

高橋 桃子*; 鹿園 直哉

Takahashi, Momoko*; Shikazono, Naoya

We constructed lesion-containing plasmids using a novel approach in order to analyze repair of clustered DNA damage. Single stranded circular DNA (the 1st strand) was synthesized in vitro from DNA oligonucleotide as a primer. The template, which is single-stranded circular DNA containing uracil, was then digested with UDG and nucleases. From the resulting 1st strand, the 2nd strand was again synthesized in vitro from a complementary DNA oligonucleotide. As DNA lesions could be placed at any position in the DNA oligonucleotide on either strand, the method allows us to construct a clustered DNA damage site in a plasmid. We have confirmed that the double-stranded plasmid DNA could be constructed with DNA lesions. The constructed plasmid would be highly useful in analyzing in vivo processing of clustered DNA damage. The level of repair of lesions within clustered DNA damage in E. coli is discussed.