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Updated: Aug 8, 2026
Continuous Fluorescence-Based Endonuclease-Coupled DNA Methylation Assay to Screen for DNA Methyltransferase Inhibitors
Published on: August 5, 2022
Functional studies of the small subunit of EcoHK31I DNA methyltransferase
Wai-To Fung1, Kong-Hung Sze, Kai-Fai Lee
1Department of Biochemistry and Centre for Protein Science and Crystallography, The Chinese University of Hong Kong, Shatin, NT, Hong Kong, China.
Abstract:
EcoHK31I DNA methyltransferase recognizes the sequence 5'-YGGCCR-3' and adds a methyl group to the fifth position of the internal cytosine to protect the DNA from cleavage by its cognate endonuclease. M.EcoHK31I is composed of polypeptides alpha and beta. Polypeptide beta only contains the conserved IX motif of the C5-MTase family, and provides a unique example to show that this motif alone may be dislocated to another polypeptide. By electromobility shift assay, we found that the alpha/beta complex recognizes specific oligonucleotide substrates. Polypeptide alpha formed aggregates with DNA, while polypeptide beta alone did not bind DNA. Therefore, polypeptide beta assists in the proper binding of polypeptide alpha to DNA substrate. The complex of polypeptide alpha and a polypeptide beta variant with an N-terminal deletion of 41 amino acids showed a 16-fold reduction in methylation activity. Further deletion resulted in an inactive methyltransferase. The dissociation equilibrium constant (Kd) of the alpha/beta complex was 56.4 nM, while the Kd value for the alpha/deltaN46-polypeptide beta complex was increased approximately 95-fold, caused by a drastic decrease in dissociate rate constant (kd) and an increase in the association rate constant (ka). This indicates that the N-terminal region of polypeptide beta takes part in subunit interaction, while the C-terminal region is involved in DNA binding.
Insights
EcoHK31I DNA methyltransferase, composed of alpha and beta polypeptides, requires both for specific DNA binding and methylation. Polypeptide beta is crucial for alpha
Area of Science:
- Molecular Biology
- Enzymology
- DNA Methylation
Background:
- EcoHK31I DNA methyltransferase methylates the 5'-YGGCCR-3' sequence, protecting DNA from its cognate endonuclease.
- The enzyme comprises two subunits, alpha and beta, with polypeptide beta uniquely containing only the conserved C5-MTase IX motif.
Purpose of the Study:
- To investigate the functional roles of the alpha and beta polypeptides in M.EcoHK31I activity.
- To elucidate the contribution of polypeptide beta's N-terminal region and C-terminal region to enzyme function.
Main Methods:
- Electromobility shift assays (EMSA) were employed to study DNA-protein interactions.
- Methylation activity assays were performed using variants of polypeptide beta with N-terminal deletions.
- Kinetic parameters, including dissociation equilibrium constant (Kd), association rate constant (ka), and dissociation rate constant (kd), were determined.
Main Results:
- The alpha/beta complex specifically recognizes oligonucleotide substrates, while polypeptide alpha alone aggregates with DNA and polypeptide beta alone does not bind DNA.
- N-terminal deletions in polypeptide beta significantly reduced methylation activity, with a 41-amino acid deletion causing a 16-fold reduction.
- The N-terminal region of polypeptide beta is critical for subunit interaction, and its C-terminal region is involved in DNA binding.
Conclusions:
- Polypeptide beta is essential for the proper binding of polypeptide alpha to the DNA substrate, acting as a crucial interaction partner.
- The N-terminal region of polypeptide beta mediates subunit interactions, while its C-terminal region directly participates in DNA binding.
- The study highlights a unique mechanism where a single conserved motif in one polypeptide assists another in DNA binding and enzymatic activity.
