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Structure-Function Differences Among Type I Restriction Endonuclease

Molecular Mass of Enzymes and Proposed Stoichiometry

The stoichiometry of R.EcoKI restriction endonuclease is know to be R2M2S1. The DNA methyltransferase component of the endonuclease is know to have a stoichiometry of M2S1. However several pieces of information point to R.EcoR124I having an unusual stoichiometry: 

Enzyme        Molecular     Ratio of         Proposed

              Mass (1)      Subunit          Stoichiometry

             (Daltons)     Densities (2)

                           HsdR  HsdM  HsdS

HsdR+MTase       -         4.4   2.3   1

(2:1)

HsdR+MTase       -         2.5   2.3   1

(1:1)

R.EcoR124I    312,000      2.6   2.1   1       R1M2S1

MTase(D50)    167,000       -    2     1(3)    M2DS2

ENase(D50)    322,000      2.4   1.9   1(4)    R1M2DS2
  1. The molecular mass of proteins was measured by size exclusion chromatography
  2. SDS-PAGE protein gels with a range of loadings of enzyme preparations and HsdR+MTase mixtures (2:1 and 1:1) were stained with Coomassie Blue and quantified by scanning. data corresponding to a loading of ~7pmol of protein was used.
  3. Data for HsdS(D50) - a deletion mutant of HsdS.
  4. Sum of the data for HsdS(D50) and HsdS(D50)' - see gel above.

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© University of Portsmouth
Author Dr Keith Firman,
Page last updated September 28, 2008