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Updated: Aug 15, 2026

DNA Methylation: Bisulphite Modification and Analysis
12:34

DNA Methylation: Bisulphite Modification and Analysis

Published on: October 21, 2011

The effect of methylation outside the recognition sequence of restriction endonuclease PvuII on its cleavage

Q Liu1, X Chen, X Zhao

  • 1Institute for Molecular Biology, Nankai University, Tianjin, People's Republic of China.

Gene
|April 1, 1992
PubMed

Insights

DNA methylation by Dam and Dcm enzymes outside recognition sites significantly impacts PvuII restriction enzyme activity. This finding is crucial for understanding DNA cleavage kinetics and epigenetic modifications.

Area of Science:

  • Molecular Biology
  • Epigenetics
  • Enzymology

Background:

  • Dam and Dcm methylation are epigenetic modifications affecting DNA.
  • Previous studies indicated methylation outside recognition sites can inhibit enzyme cleavage.
  • The PvuII restriction enzyme is sensitive to DNA methylation patterns.

Purpose of the Study:

  • To investigate the effect of Dam and Dcm methylation on PvuII restriction enzyme activity at specific sites.
  • To determine if methylation outside the PvuII recognition sequence influences cleavage efficiency.
  • To characterize the kinetic differences in PvuII cleavage between methylated and unmethylated DNA.

Main Methods:

  • Construction of a new recombinant plasmid (pGEM4-SV40ori-anti-ras) with two PvuII sites.
  • Production of both methylated (Dam and Dcm) and unmethylated plasmid DNA in Escherichia coli.
  • Linearization of DNA using ScaI restriction enzyme.
  • Digestion of DNA with varying concentrations of PvuII enzyme.
  • Analysis of DNA fragments using agarose gel electrophoresis to assess cleavage rates.

Main Results:

  • PvuII site I on methylated DNA was cleaved 4- to 8-fold slower than site II.
  • In unmethylated DNA, both PvuII sites were cleaved at comparable rates.
  • The observed difference in cleavage rates was solely attributed to Dam and Dcm methylation outside the PvuII recognition sequences.
  • A Dam methylation site adjacent to PvuII site I likely caused the inhibition.

Conclusions:

  • Dam and Dcm methylation outside recognition sequences significantly inhibit PvuII enzyme activity.
  • The position of methylation relative to the recognition site is critical for its inhibitory effect.
  • A new parameter considering methylation outside recognition sites should be incorporated into DNA cleavage kinetic studies.

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