Recognition of an Unnatural Difluorophenyl Nucleotide by Uracil DNA Glycosylase

Abstract

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.

Examination of the Long-range Effects of Aminofluorene-induced Conformational Heterogeneity and Its Relevance to the Mechanism of Translesional DNA Synthesis

“…The lower limit for k C was determined to be 1715 s −1 , which is much faster than the rates of spontaneous base pair opening in a regular B-DNA molecule (44,45). The situation resembles base flipping by the DNA repair enzyme uracil DNA glycosylase (46).…”

Bacterial cell wall composition and the influence of antibiotics by cell-wall and whole-cell NMR

“…Structural studies have been performed with antibiotics bound to cell walls or whole cells, typically prepared in trehalose-or ethanolamine-based lyophilization buffers, wherein trehalose serves as an effective hydrogen bonding partner as bulk water is removed during lyophilization. Such lyophilized formulations have permitted structure-based studies of enzymes such as lumazine synthase [42], EPSP synthase [43], factor Xa [44] and uracil DNA glycosylase [45], and are not perturbative to structure. The enzymes can maintain their enzymatic activity even after NMR measurements upon sample rehydration and assay.…”

Engineered Extrahelical Base Destabilization Enhances Sequence Discrimination of DNA Methyltransferase M.HhaI

“…In spite of extensive kinetic and structural investigations, the mechanisms of base-flipping and base stabilization, and their importance to rate enhancements and specificity generation, remain ambiguous. [24][25][26][27] We have shown that Val121 in M.HhaI, positioned immediately above the flipped-out cytosine ( Figure 1(b)), is extremely important for initiating and/or maintaining the flipped state. 23 Replacement of Val121 with alanine results in a 10 5 -fold loss of DNA affinity, which is recovered entirely when the mutant is presented with DNA lacking a base at the target cytosine position.…”