Recognition of an unnatural difluorophenyl nucleotide by uracil DNA glycosylase

Lin Jiang Yu, Lynda M. McDowell, Barbara Poliks, Daniel R. Studelska, Chunyang Cao, Gregory S. Potter, Jacob Schaefer, Fenhong Song, James T. Stivers

Research output: Contribution to journalArticlepeer-review

28 Scopus citations


The DNA repair enzyme uracil DNA glycosylase (UDG) utilizes base flipping to recognize and remove unwanted uracil bases from the genome but does not react with its structural congener, thymine, which differs by a single methyl group. Two factors that determine whether an enzyme flips a base from the duplex are its shape and hydrogen bonding properties. To probe the role of these factors in uracil recognition by UDG, we have synthesized a DNA duplex that contains a single difluorophenyl (F) nucleotide analogue that is an excellent isostere of uracil but possesses no hydrogen bond donor or acceptor groups. By using binding affinity measurements, solution 19F NMR, and solid state 31P{19F} rotational-echo double-resonance (REDOR) NMR measurements, we establish that UDG partially unstacks F from the duplex. However, due to the lack of hydrogen bonding groups that are required to support an open-to-closed conformational transition in UDG, F cannot stably dock in the UDG active site. We propose that F attains a metastable unstacked state that mimics a previously detected intermediate on the uracil-flipping pathway and suggest structural models of the metastable state that are consistent with the REDOR NMR measurements.

Original languageEnglish (US)
Pages (from-to)15429-15438
Number of pages10
Issue number49
StatePublished - Dec 14 2004

ASJC Scopus subject areas

  • Biochemistry


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