Recognition of an Unnatural Difluorophenyl Nucleotide by Uracil DNA Glycosylase

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• 作者：Yu Lin Jiang ; Lynda M. McDowell ; Barbara Poliks ; Daniel R. Studelska ; Chunyang Cao ; Gregory S. Potter ; Jacob Schaefer ; Fenhong Song ; and James T. Stivers
• 刊名：Biochemistry
• 出版年：2004
• 出版时间：December 14, 2004
• 年：2004
• 卷：43
• 期：49
• 页码：15429 - 15438
• 全文大小：277K
• 年卷期：v.43,no.49(December 14, 2004)
• ISSN：1520-4995

文摘

The DNA repair enzyme uracil DNA glycosylase (UDG) utilizes base flipping to recognizeand 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 fromthe duplex are its shape and hydrogen bonding properties. To probe the role of these factors in uracilrecognition by UDG, we have synthesized a DNA duplex that contains a single difluorophenyl (F) nucleotideanalogue 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-closedconformational transition in UDG, F cannot stably dock in the UDG active site. We propose that F attainsa metastable unstacked state that mimics a previously detected intermediate on the uracil-flipping pathwayand suggest structural models of the metastable state that are consistent with the REDOR NMRmeasurements.