Related Experiment Videos
DNA binding and subunit interactions in the type I methyltransferase M.EcoR124I
D R Mernagh1, L A Reynolds, G G Kneale
1Biophysics Laboratories, School of Biological Sciences, University of Portsmouth, St Michael's Building, Portsmouth PO1 2DT, UK.
Nucleic Acids Research
|March 1, 1997
Summary
The HsdM subunit is crucial for DNA binding in the M.EcoR124I DNA methyltransferase, even when the HsdS subunit is fused to GST. Removing GST restores DNA binding and methylation activity.
Area of Science:
- Molecular Biology
- Enzymology
- Epigenetics
Background:
- The type I DNA methyltransferase M.EcoR124I comprises HsdM (methylation) and HsdS (recognition) subunits.
- HsdS is insoluble independently but soluble as a GST fusion protein.
Purpose of the Study:
- To investigate the roles of HsdM and HsdS subunits in M.EcoR124I complex formation and DNA methylation.
- To determine the impact of GST fusion on HsdS function and DNA binding.
Main Methods:
- Expression and purification of HsdS as a GST fusion protein.
- Complex formation assays with HsdM and HsdS-GST.
- DNA binding and methylation activity assays.
- GST removal to assess subunit function.
Main Results:
- HsdS-GST alone did not form discrete complexes with DNA.
- HsdM addition to HsdS-GST formed complexes but lacked methylation activity.
- Complex formation suggested HsdM binding sites on HsdS were affected by GST.
- GST removal restored specific DNA binding and full methylation activity.
Conclusions:
- The HsdM subunit is essential for DNA binding, not just catalysis.
- The N-terminus of HsdS is important for HsdM interaction and complex integrity.
- GST fusion inhibits DNA binding, highlighting the importance of an accessible HsdS N-terminus for proper enzyme function.