Abstract
DNA cytosine-5-methyltransferase HhaI recognizes the GCGC sequence and flips the inner cytosine out of DNA helix and into the catalytic site for methylation. The 5'-phosphate of the flipped out cytosine is in contact with the conserved Thr-250 from the target recognition domain. We have produced 12 mutants of Thr-250 and examined their methylation potential in vivo. Six active mutants were subjected to detailed biochemical and structural studies. Mutants with similar or smaller side chains (Ser, Cys, and Gly) are very similar to wild-type enzyme in terms of steady-state kinetic parameters k(cat), K(m)(DNA), K(m)(AdoMet). In contrast, the mutants with bulkier side chains (Asn, Asp, and His) show increased K(m) values for both substrates. Fluorescence titrations and stopped-flow kinetic analysis of interactions with duplex oligonucleotides containing 2-aminopurine at the target base position indicate that the T250G mutation leads to a more polar but less solvent-accessible position of the flipped out target base. The x-ray structure of the ternary M. HhaI(T250G).DNA.AdoHcy complex shows that the target cytosine is locked in the catalytic center of enzyme. The space created by the mutation is filled by water molecules and the adjacent DNA backbone atoms dislocate slightly toward the missing side chain. In aggregate, our results suggest that the side chain of Thr-250 is involved in constraining the conformation the DNA backbone and the target base during its rotation into the catalytic site of enzyme.
Highlights
Recognition and modification of specific residues in DNA is a key event in many cellular processes
The target base populates multiple conformational states spanning from its original position in the DNA helix to its final position in the catalytic pocket
Analysis of the base-flipping capability of the mutants in the presence of a fluorescent DNA substrate shows that the initial base-unstacking phase is similar in all cases, subsequent behavior of the binary complex is markedly different for the T250G and T250D mutants
Summary
Mutagenesis—Restriction endonucleases, DNA modification enzymes and kits were obtained from MBI Fermentas. Exact concentrations were determined by active site titration with a 37-mer fluorescent duplex oligonucleotide (see bellow) or 32P-labeled hemimethylated 37mer duplex in the presence of AdoHcy (not shown) and the values amounted to ϳ60% of those obtained in the Coomassie measurements. Fluorescence intensity measurements were performed at 25 °C on an SLM-Aminco AB2 spectrofluorimeter at excitation wavelength (Ex) of 320 nm and emission wavelength (Em) of 381 nm as described previously [14]. Titration of 100 nM 37-mer GPGC/GMGC duplex was performed by incremental addition of 4 M M.HhaI in Reaction buffer 200 nM GPGC/GMGC in Reaction buffer was titrated by incremental addition of 25 M M.HhaI until saturation of fluorescence level was achieved. Crystallography—The structure of the T250G mutant of M.HhaI in a ternary complex with AdoHcy and a short DNA duplex was determined by x-ray crystallography (Protein Data Bank entry code 1FJX). After several rounds of least square refinement, manual model building, and placement of well ordered solvent molecules (interpreted as water) by examination of difference electron density, the crystallographic R-free and R-factor (ϭ͉Fo Ϫ Fc/͚ ͉Fo, where Fo and Fc are the observed and calculated structure factor amplitudes, respectively) were reduced to 28.3% for 1,896 (approximately 8% of total unique reflections) and 20.7% for 22,169 unique reflections, respectively
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