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Sequence-specific Labeling of Nucleic Acids and Proteins with Methyltransferases and Cofactor Analogues
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Methyl-specific DNA binding by McrBC, a modification-dependent restriction enzyme

F J Stewart1, D Panne, T A Bickle

  • 1New England Biolabs, Inc, Beverly, MA 01915, USA.

Journal of Molecular Biology
|May 2, 2000
PubMed

Insights

The endonuclease McrBC (Methylated-CpG recognition protein BC) binds specifically to methylated DNA, requiring GTP. McrC influences complex formation without altering the DNA-protein interface, suggesting a conformational role.

Area of Science:

  • Molecular Biology
  • Biochemistry
  • Genetics

Background:

  • McrBC is a modification-dependent endonuclease in Escherichia coli K-12.
  • It recognizes methylated DNA sites (5' R(m)C 3') and requires GTP for activity.
  • DNA cleavage typically involves coordination between two recognition sites via translocation.

Purpose of the Study:

  • To investigate the assembly of the McrBC cleavage-competent complex.
  • To elucidate the roles of McrB and McrC in DNA binding and complex formation.
  • To understand the mechanism of McrBC-mediated DNA cleavage.

Main Methods:

  • Gel-shift assays to analyze McrB(L) binding to methylated DNA.
  • DNase I footprinting to determine DNA-protein interactions and cooperativity.
  • Competition assays using single-stranded DNA to assess binding specificity.

Main Results:

  • McrB(L) binding is specific for methylated DNA, requires GTP and Mg(2+), and is sequence-dependent.
  • Single-stranded DNA competes for binding in a modification- and sequence-specific manner.
  • McrC and GTPgammaS induce a supershifted complex; DNase I footprinting shows modest cooperativity and protection of spacer DNA, with McrC not altering the footprint.

Conclusions:

  • McrBC assembly involves specific binding of McrB(L) to methylated DNA.
  • McrC appears to induce a conformational change in the McrBC-DNA complex rather than reorganizing the DNA-protein interface.
  • These findings provide insights into the mechanism of modification-dependent DNA cleavage by McrBC.

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