Rational engineering of a DNA glycosylase specific for an unnatural cytosine:pyrene base pair.
A novel site-specific cytosine DNA glycosylase has been rationally engineered from the active site scaffold of the DNA repair enzyme uracil DNA glycosylase (UDG). UDG, which operates by a nucleotide flipping mechanism, was first converted into a sequence nonspecific cytosine DNA glycosylase (CDG) by altering the base-specific hydrogen bond donor-acceptor groups in the active site. A second mutation that renders UDG defective in nucleotide flipping was then introduced, and the double mutant was rescued using a substrate with a "preflipped" cytosine base. Substrate-assisted flipping was engineered by incorporation of an unnatural pyrene nucleotide wedge (Y) into the DNA strand opposite to the target cytosine. This new enzyme, CYDG, can be used to target cleavage of specific cytosine residues in the context of a C/Y base pair in any DNA fragment.
Department of Pharmacology and Molecular Sciences, The Johns Hopkins University School of Medicine, 725 North Wolfe Street, Baltimore, MD 21205, USA.
Keehwan
K
Kwon
Yu Lin
YL
Jiang
James T
JT
Stivers
D000465:Algorithms; D020029:Base Pairing; D001483:Base Sequence; D002384:Catalysis; D003596:Cytosine / Q000737:chemistry; D045647:DNA Glycosylases / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics; D004260:DNA Repair; D018076:DNA, Complementary / Q000235:genetics; D005804:Genes, Lethal / Q000235:genetics; D005818:Genetic Engineering; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008969:Molecular Sequence Data; D011721:Pyrenes / Q000737:chemistry; D013379:Substrate Specificity
D018076:DNA, Complementary; D011721:Pyrenes; D003596:Cytosine; D045647:DNA Glycosylases