Year-end Sale % OFF 
Abstract
Base flipping is a highly conserved process by which enzymes swivel an entire nucleotide from the DNA base stack into their active site pockets. Uracil DNA glycosylase (UDG) is a paradigm enzyme that uses a base flipping mechanism to catalyze the hydrolysis of the N-glycosidic bond of 2'-deoxyuridine (2'-dUrd) in DNA as the first step in uracil base excision repair. Flipping of 2'-dUrd by UDG has been proposed to follow a "pushing" mechanism in which a completely conserved leucine side chain (Leu-191) is inserted into the DNA minor groove to expel the uracil. Here we report a novel implementation of the "chemical rescue" approach to show that the weak binding affinity and low catalytic activity of L191A or L191G can be completely or partially restored by substitution of a pyrene (Y) nucleotide wedge on the DNA strand opposite to the uracil base (U/A to U/Y). These results indicate that pyrene acts both as a wedge to push the uracil from the base stack in the free DNA and as a "plug" to hinder its reinsertion after base flipping. Pyrene rescue should serve as a useful and novel tool to diagnose the functional roles of other amino acid side chains involved in base flipping.
Highlights
A remarkable and evolutionarily conserved aspect of enzymatic recognition of damaged bases in DNA is the process of base flipping [1]
Uracil DNA glycosylase (UDG) is a paradigm enzyme that uses a base flipping mechanism to catalyze the hydrolysis of the Nglycosidic bond of 2-deoxyuridine (2-dUrd) in DNA as the first step in uracil base excision repair
DNA glycosylases, which catalyze the first step in DNA base excision repair, are one general enzyme class that must act through a base flipping mechanism [2]
Summary
Oligonucleotide Synthesis—The substrates and substrate mimics were synthesized using standard phosphoramidite chemistry with an Applied Biosystems 390 synthesizer. The DNA strands were hybridized as previously described to form the duplexes used in the kinetic and binding studies as shown in Table I [8]. In these sequences, P ϭ 2-aminopurine deoxynucleotide, U ϭ 2Ј-fluoro-2Ј-deoxyuridine nucleotide, and Y ϭ pyrene deoxyribonucleotide. For the 2-aminopurine (2-AP) and pyrene-labeled molecules, direct fluorescence binding measurements were made by titrating fixed concentrations of the DNA with increasing amounts of UDG. Molecular Modeling—A model for the pyrene nucleotide in the context of a duplex DNA bound to L191A UDG was generated from the crystal coordinates of the ternary complex of human UDG bound to the products uracil and abasic DNA (Protein Data Bank entry 1SSP). The corresponding leucine side chain was mutated computationally to ala-
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
