Abstract
Nucleotide excision repair (NER) is a general DNA repair mechanism that is capable of removing a wide variety of DNA lesions induced by physical or chemical insults. UvrD, a member of the helicase SF1 superfamily, plays an essential role in bacterial NER by unwinding the duplex DNA in the 3' to 5' direction to displace the lesion-containing strand. In order to achieve conditional control over NER, we generated a light-activated DNA helicase. This was achieved through a site-specific incorporation of a genetically encoded hydroxycoumarin lysine at a crucial position in the ATP-binding pocket of UvrD. The resulting caged enzyme was completely inactive in several functional assays. Moreover, enzymatic activity of the optically triggered UvrD was comparable to that of the wild-type protein, thus demonstrating excellent OFF to ON switching of the helicase. The developed approach provides optical control of NER, thereby laying a foundation for the regulation of ATP-dependent helicase functions in higher organisms. In addition, this methodology is applicable to the light-activation of a wide range of ATPases.