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Protein fragment complementation in M.HhaI DNA methyltransferase
Wonchae Choe1, Srinivasan Chandrasegaran, Marc Ostermeier
1Department of Chemical and Biomolecular Engineering, Johns Hopkins University, 3400 N. Charles St. Baltimore, MD 21218, USA.
Biochemical and Biophysical Research Communications
|July 26, 2005
Summary
Researchers split the 5-methylcytosine (5mC) DNA methyltransferase M.HhaI into two inactive fragments. These fragments can reassemble in vivo to form an active enzyme, enabling efficient DNA methylation.
Area of Science:
- Enzymology
- Molecular Biology
- Epigenetics
Background:
- The 5-methylcytosine (5mC) DNA methyltransferase M.HhaI is crucial for DNA methylation.
- M.HhaI can be functionally reconstituted from two individually inactive N- and C-terminal fragments.
Purpose of the Study:
- To identify and characterize split versions of the M.HhaI methyltransferase.
- To investigate protein fragment complementation for creating active DNA methyltransferases.
Main Methods:
- Construction and screening of M.HhaI gene fragment libraries.
- Selection of fragment pairs capable of forming an active 5mC methyltransferase in vivo.
- Identification of the bisection site within the M.HhaI sequence.
Main Results:
- An active pair of M.HhaI fragments was successfully identified through library screening.
- The optimal site for splitting M.HhaI was found in the variable region near the target recognition domain (TRD), between motif VIII and the TRD.
- This bisection site is conserved in other methyltransferases like M.AquI and engineered variants of M.BspRI.
Conclusions:
- Protein fragment complementation is a viable strategy for reconstituting the activity of split DNA methyltransferases.
- The identified split site in M.HhaI provides insights into enzyme structure-function relationships and engineering possibilities.