title: Optical Control of DNA Helicase Function through Genetic Code Expansion.
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.
authors.affiliation: Department of Chemistry, University of Pittsburgh, Pittsburgh, Pennsylvania, 15260, USA.
authors.forename: Ji
authors.initials: J
authors.lastname: Luo
authors.affiliation: Department of Pharmacology and Chemical Biology, USA.|The University of Pittsburgh Cancer Institute, Hillman Cancer Center, Pittsburgh, Pennsylvania, 15213, USA.
authors.forename: Muwen
authors.initials: M
authors.lastname: Kong
authors.affiliation: The University of Pittsburgh Cancer Institute, Hillman Cancer Center, Pittsburgh, Pennsylvania, 15213, USA.
authors.forename: Lili
authors.initials: L
authors.lastname: Liu
authors.affiliation: Department of Chemistry, University of Pittsburgh, Pittsburgh, Pennsylvania, 15260, USA.
authors.forename: Subhas
authors.initials: S
authors.lastname: Samanta
authors.affiliation: Department of Pharmacology and Chemical Biology, USA.|The University of Pittsburgh Cancer Institute, Hillman Cancer Center, Pittsburgh, Pennsylvania, 15213, USA.
authors.forename: Bennett
authors.initials: B
authors.lastname: Van Houten
authors.affiliation: Department of Chemistry, University of Pittsburgh, Pittsburgh, Pennsylvania, 15260, USA.|The University of Pittsburgh Cancer Institute, Hillman Cancer Center, Pittsburgh, Pennsylvania, 15213, USA.
authors.forename: Alexander
authors.initials: A
authors.lastname: Deiters
mesh_terms: D001419:Bacteria / Q000201:enzymology; D004265:DNA Helicases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D004260:DNA Repair / Q000235:genetics / Q000502:physiology*; D004789:Enzyme Activation / Q000528:radiation effects; D005453:Fluorescence; D005815:Genetic Code; D008027:Light; D015394:Molecular Structure; D015202:Protein Engineering
chemical_list: D004265:DNA Helicases
keywords: DNA damage; caged compounds; helicase; light activation; unnatural amino acids