Recognition of an unnatural difluorophenyl nucleotide by uracil DNA glycosylase.
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 (19)F NMR, and solid state (31)P[(19)F] 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.
Jiang
Yu Lin
YL
Department of Pharmacology and Molecular Sciences, The Johns Hopkins University School of Medicine, 725 North Wolfe Street, Baltimore, Maryland 21205-2185, USA.
McDowell
Lynda M
LM
Poliks
Barbara
B
Studelska
Daniel R
DR
Cao
Chunyang
C
Potter
Gregory S
GS
Schaefer
Jacob
J
Song
Fenhong
F
Stivers
James T
JT
D019943:Amino Acid Substitution; D001665:Binding Sites / Q000235:genetics; D002384:Catalysis; D045647:DNA Glycosylases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004795:Enzyme Stability; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D005467:Floxuridine / Q000138:chemical synthesis / Q000378:metabolism*; D005461:Fluorine / Q000378:metabolism; D006860:Hydrogen Bonding; D007930:Leucine / Q000737:chemistry / Q000235:genetics; D008958:Models, Molecular; D019906:Nuclear Magnetic Resonance, Biomolecular; D009692:Nucleic Acid Heteroduplexes / Q000138:chemical synthesis / Q000235:genetics / Q000378:metabolism; D012996:Solutions; D013050:Spectrometry, Fluorescence; D013379:Substrate Specificity; D013816:Thermodynamics; D014498:Uracil / Q000737:chemistry / Q000378:metabolism*; D051981:Uracil-DNA Glycosidase
D029968:Escherichia coli Proteins; D009692:Nucleic Acid Heteroduplexes; D012996:Solutions; D005467:Floxuridine; D005461:Fluorine; D014498:Uracil; C037289:2'fluoro-2'-deoxyuridine; D045647:DNA Glycosylases; D051981:Uracil-DNA Glycosidase; D007930:Leucine