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Updated: Aug 6, 2026
Atomic Force Microscopy Investigations of DNA Lesion Recognition in Nucleotide Excision Repair
Published on: May 24, 2017
The McrBC endonuclease translocates DNA in a reaction dependent on GTP hydrolysis
D Panne1, E A Raleigh, T A Bickle
1Department of Microbiology, Biozentrum, Basel University, Klingelbergstrasse 70, Basel, CH-4056, Switzerland.
Abstract:
McrBC specifically recognizes and cleaves methylated DNA in a reaction dependent on GTP hydrolysis. DNA cleavage requires at least two recognition sites that are optimally separated by 40-80 bp, but can be spaced as far as 3 kb apart. The nature of the communication between two recognition sites was analyzed on DNA substrates containing one or two recognition sites. DNA cleavage of circular DNA required only one methylated recognition site, whereas the linearized form of this substrate was not cleaved. However, the linearized substrate was cleaved if a Lac repressor was bound adjacent to the recognition site. These results suggest a model in which communication between two remote sites is accomplished by DNA translocation rather than looping. A mutant protein with defective GTPase activity cleaved substrates with closely spaced recognition sites, but not substrates where the sites were further apart. This indicates that McrBC translocates DNA in a reaction dependent on GTP hydrolysis. We suggest that DNA cleavage occurs by the encounter of two DNA-translocating McrBC complexes, or can be triggered by non-specific physical obstacles like the Lac repressor bound on the enzyme's path along DNA. Our results indicate that McrBC belongs to the general class of DNA "motor proteins", which use the free energy associated with nucleoside 5'-triphosphate hydrolysis to translocate along DNA.
Insights
McrBC (Methylated CpG restriction protein C) enzyme cleaves methylated DNA by translocating along it, a process requiring GTP hydrolysis. This DNA motor protein
Area of Science:
- Molecular Biology
- Enzymology
- DNA-protein interactions
Background:
- McrBC is an enzyme that cleaves methylated DNA.
- This cleavage is dependent on GTP hydrolysis and requires specific DNA recognition sites.
Purpose of the Study:
- To investigate the mechanism of communication between McrBC recognition sites on DNA.
- To determine if McrBC translocates DNA or communicates via DNA looping.
- To elucidate the role of GTP hydrolysis in McrBC function.
Main Methods:
- Analysis of DNA substrates with one or two methylated recognition sites.
- Use of circular and linearized DNA substrates.
- Employing a mutant McrBC protein with defective GTPase activity.
- Utilizing Lac repressor as a physical obstacle on DNA.
Main Results:
- DNA cleavage of circular DNA required only one methylated recognition site, while linearized DNA required additional factors.
- Linearized DNA was cleaved when a Lac repressor was bound near the recognition site.
- A mutant McrBC protein with defective GTPase activity could cleave DNA with closely spaced sites but not distant sites.
- These findings support a DNA translocation model over DNA looping for McrBC function.
Conclusions:
- McrBC functions as a DNA motor protein, translocating along DNA using GTP hydrolysis.
- Communication between distant recognition sites is achieved through DNA translocation.
- DNA cleavage is likely triggered by the encounter of two translocating McrBC complexes or physical obstacles.
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