Point mutations in the central conserved region of the DNA recognition subunit of R.EcoR124I affecting subunit assembly of the endonuclease

Dr. Marie Weiserova and Keith Firman

Dr Keith Firman, Biophysics Laboratories, School of Biological Sciences, University of Portsmouth, St. Michael's Building, White Swan Road, Portsmouth, PO1 2DT, United Kingdom

Dr Marie Weiserova, Institute of Microbiology, Czech Academy of Sciences, Videnska 1083, 14240 Prague 4, Czech Republic

We have used deletion mutagenesis and PCR-based misincorporation mutagenesis to produce a collection of mutations in the central conserved region of the DNA binding subunit of the type IC restriction endonuclease EcoR124I.  It has been proposed that this domain is involved in protein-protein interactions during the assembly of the endonuclease.  While a large percentage of these mutations gave a classical Res^- Mod^- phenotype, one mutant was isolated with a non-classical Res^- Mod⁺ phenotype.  The loss of restriction activity, but retention of the ability to modify indicates that this mutation cannot affect DNA binding and must alter the assembly of the endonuclease in such a way as to prevent DNA cleavage but allow methylation.  This mutant resulted from a single amino acid change Trp²¹²Arg.  The location of the single amino acid change is at the border of the central conserved region and the second target recognition domain (TRD2) and suggests that this region is extremely important for the assembly of the methylase with HsdR subunit into a functional restriction endonuclease.

1. Abstract
2. Results
3. Figure 1
4. Figure 2
5. Figure 3
6. Conclusions

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