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Related Experiment Video

Updated: Jul 12, 2026

Sequence-specific Labeling of Nucleic Acids and Proteins with Methyltransferases and Cofactor Analogues

Sequence-specific Labeling of Nucleic Acids and Proteins with Methyltransferases and Cofactor Analogues

Published on: November 22, 2014

Sequence-specific DNA binding by the MspI DNA methyltransferase.

A K Dubey¹, R J Roberts

¹Cold Spring Harbor Laboratory, NY 11724.

Nucleic Acids Research

|June 25, 1992

Summary

The MspI methyltransferase binds DNA specifically, with highest affinity to hemimethylated sequences when S-adenosyl-L-homocysteine is present. Cofactors significantly enhance sequence discrimination.

Area of Science:

Molecular Biology
Enzymology
Epigenetics

Background:

MspI methyltransferase (M.MspI) modifies DNA at the CCGG sequence, creating 5-methylcytosine.
Understanding DNA-binding properties is crucial for enzyme function.

Purpose of the Study:

Investigate the sequence-specific DNA-binding properties of M.MspI.
Determine binding affinities under various conditions, including cofactor presence.

Main Methods:

Gel-mobility shift assays to assess binding equilibrium.
DNaseI footprinting to map DNA-protein interactions.

Main Results:

M.MspI exhibits sequence-specific DNA binding with or without methyl donors (S-adenosyl-L-methionine, sinefungin, S-adenosyl-L-homocysteine).

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Highest affinity observed for hemimethylated DNA (Kd = 3.6 x 10(-7) M) with S-adenosyl-L-homocysteine.

Specific binding to unmethylated and fully methylated DNA was also observed, albeit with lower affinity.

Cofactors increased discrimination between specific and non-specific DNA sequences up to 100-fold.

DNaseI footprinting revealed M.MspI covers 16 base pairs, with the CCGG site asymmetrically positioned.

Conclusions:

M.MspI binding affinity is modulated by DNA methylation status and the presence of cofactors.
Cofactors significantly enhance the specificity of M.MspI for its recognition site.
The enzyme displays a preference for hemimethylated DNA, suggesting roles in DNA repair or replication.