Backbone Conformational Equilibrium in Mismatched DNA Correlates with Enzyme Activity

Area of Science:

• Biochemistry
• Molecular Biology
• Genetics

Background:

• T:G mismatches in DNA are primarily caused by methylated CpG deamination or incorrect nucleotide incorporation.
• The mechanism by which DNA repair enzymes recognize these mismatches is not fully understood.
• Protein-DNA backbone interactions are hypothesized to play a crucial role in mismatch recognition and repair.

Purpose of the Study:

• To investigate the role of DNA phosphate backbone energetics in mismatch recognition.
• To correlate DNA backbone equilibrium with the substrate dependence of various DNA repair enzymes.
• To elucidate the conformational properties facilitating protein-DNA interactions during repair.

Main Methods:

• Utilized 31P Nuclear Magnetic Resonance (NMR) spectroscopy to study DNA backbone BI-BII interconversion energetics.
• Compared energetic differences (ΔG) between canonical DNA, mismatched DNA, and DNA with lesions.
• Correlated DNA phosphate backbone equilibrium (Keq) with enzyme kinetics and binding parameters for thymine DNA glycosylase (TDG), MBD4, and other enzymes.

Main Results:

• Previously found stepwise differences in ΔG (1-2 kcal/mol) for backbone interconversion in modified DNA compared to canonical DNA.
• Established strong correlations between DNA phosphate backbone equilibrium (Keq) and the kinetics/binding parameters of tested enzymes.
• Demonstrated sequence and base-pair dependence in these correlations.

Conclusions:

• DNA phosphate backbone equilibrium is strongly correlated with the substrate dependence of key DNA repair enzymes.
• This backbone equilibrium likely plays a significant role in mismatch recognition.
• Conformational rearrangements and energetics during enzyme interrogation, such as nucleotide flipping, are influenced by backbone properties.