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Inhibition of MspI cleavage activity by hydroxymethylation of the CpG site: a concern for DNA modification studies

Kenji Ichiyanagi1

  • 1Division of Epigenomics, Medical Institute of Bioregulation, Kyushu University, Fukuoka, Japan. ichiyanagi@bioreg.kyushu-u.ac.jp

Epigenetics
|March 8, 2012
PubMed

Insights

The restriction enzyme MspI is inhibited by 5-hydroxymethyl-cytosine (5-hmC), affecting DNA modification studies. Alternative enzymes like TaqI and HaeIII show resistance to hydroxymethylation, suggesting careful interpretation of MspI-based analyses.

Area of Science:

  • Epigenetics
  • Molecular Biology
  • Genomics

Background:

  • Cytosine methylation in mammalian DNA, primarily at CpG sites, is a key epigenetic regulator.
  • 5-methyl-cytosine (5-mC) can be converted to 5-hydroxymethyl-cytosine (5-hmC) by ten-eleven translocation (TET) enzymes.
  • Restriction enzymes are commonly used to study DNA modifications.

Purpose of the Study:

  • To investigate the kinetic effects of 5-hydroxymethyl-cytosine (5-hmC) on the activity of the restriction enzyme MspI.
  • To compare the sensitivity of MspI, TaqI, and HaeIII to hydroxymethylation in their recognition sequences.

Main Methods:

  • Kinetic analysis of MspI activity in the presence of varying degrees of cytosine hydroxymethylation.
  • Comparative analysis of TaqI and HaeIII resistance to hydroxymethylation.

Main Results:

  • MspI activity is significantly reduced by symmetrical hydroxymethylation of its recognition site.
  • MspI activity is partially inhibited by hemi-hydroxymethylation.
  • TaqI and HaeIII exhibit relative resistance to hydroxymethylation compared to MspI.

Conclusions:

  • DNA modification studies employing MspI, including reduced representation bisulfite sequencing and 5-hmC analysis, require careful interpretation due to enzyme inhibition by 5-hmC.
  • The findings highlight the importance of considering the impact of DNA modifications on restriction enzyme activity for accurate epigenetic research.

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