Igls Poster 2 Results

Stoichiometry of Type I Restriction-Modification enzymes

Type IA

EcoKI

EcoBI

ENase is a stable complex of R₂M₂S₁
MTase forms M₂S₁ complex
M₂S₁ <--> M₁S₁ + M (K_D=70nM)

ENase is heterogeneous: R₁:M₂:S₁ , R₁M₁S₁
MTase gives M₁S₁ active complex

Type IB

EcoAI

ENase is a weak complex, when purified it dissociates to MTase and HsdR (DEAE)

Type IV

EcoR124I

MTase is a stable trimer of M₂S₁
ENase ?

It has been suggested (Janscak et al., 1996) that the stoichiometry of the purified ENase of the EcoR124I R-M system is R₁M₂S₁. However, data presented here shows that the ENase is a mixture of two stoichiometric forms.

Analysis of stoichiometry through gel retardation

Gel retardation studies of the purified EcoR124I endonuclease (ENase) showed the presence of two retarded bands, neither of which was due to non-specific binding (Figure 1, lane 2). Addition of extra HsdR shifted the lower band resulting in only a single band (Figure 1, lane 5). While addition of an excess of HsdR to EcoR124I DNA methyltransferase (MTase) also rsulted in a single upper species (Figure 1, lane 9). However, equimolar amounts of MTase and HsdR produced only the lower species (Figure 1, lane 8). Finally, addition of MTase to ENase also resulted in only the lower species (Figure 1, lane 6).

Therefore, the ENase is present as a equilibrium mixture of two species. Analysis of the ratios of HsdS, HsdM and HsdR present in each species revealed that the lower species was a complex of stoichiometry R₁M₂S₁. While the upper species has a stoichiometry of R₂M₂S₁ (Figure 2).

Figure 1

Figure 2

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