Enzyme-promoted base flipping controls DNA methylation fidelity

Area of Science:

• Biochemistry
• Molecular Biology
• Enzymology

Background:

• Enzyme catalysis and specificity rely on conformational changes, but a detailed understanding is lacking.
• The DNA cytosine methyltransferase M.HhaI serves as a model system to study these processes.

Purpose of the Study:

• To elucidate the transient states of enzyme-substrate complexes during DNA methylation.
• To investigate how conformational transitions influence the specificity and catalytic efficiency of M.HhaI.

Main Methods:

• Multidimensional, transverse relaxation-optimized nuclear magnetic resonance (NMR) spectroscopy.
• Fluorescence tracking of the catalytic loop.
• Analysis of enzyme-substrate complexes with cognate and noncognate DNA sequences.

Main Results:

• M.HhaI adopts a similar conformation with both cognate and noncognate DNA.
• Specific protein-DNA interactions facilitate target base flipping in the cognate mode.
• Noncognate substrates exhibit slow base flipping, with this transition being rate-limiting.
• Spectroscopic evidence for a previously unobserved intermediate in the base flipping pathway was obtained.

Conclusions:

• Conformational rearrangements in M.HhaI are crucial for balancing enzyme specificity and catalytic efficiency.
• The study reveals key details of the base flipping mechanism and transient states involved in DNA methylation.