The Stoichiometry of Type I REases

Type I restriction endonucleases are composed of three different subunits of which the HsdS subunit determines DNA specificity, the HsdM subunit is responsible for DNA methylation and the HsdR subunit is required for restriction. The HsdM subunit, together with HsdS subunit, can form an independent DNA methyltransferase with a stoichiometry of M₂S₁. Our gel retardation analysis of the EcoR124I restriction endonuclease, bound to a 39-mer DNA oligoduplex containing one EcoR124I recognition site, revealed that the purified protein was a mixture of two complexes forming two different specific DNA-protein complexes (Figure 1). Subunit analysis of protein isolated from both complexes (Figure 2) has shown that the larger species has a stoichiometry of R₂M₂S₁. The faster species has a stoichiometry of R₁M₂S₁ and appears to be a stable intermediate in the endonuclease assembly pathway from the trimeric methylase and HsdR subunit. Enzyme assays have shown that only the R₂M₂S₁ complex is capable of DNA cleavage (Figure 3), however, R₁M₂S₁ complex retains ATPase activity (Figure 4). Analysis of the complexes by HPLC also indicates two stoichiometric forms; although, the titration of a constant concentration of methylase with the HsdR subunit dramatically only showed the change in molecular weight of the complex upon prolonged incubation (Figure 5). Measurement of the apparent equilibrium dissociation constants of the subunit complexes has shown that the M₂S₁ and R₁M₂S₁ species are very tight complexes with Kd values lower than 10^-9 M; while the R₂M₂S₁ species is a much weaker complex, and dissociates into R₁M₂S₁ and free HsdR subunit (Figure 6).

Figure 1 Gel retardation analysis of purified EcoR124I restriction endonuclease

Lane 1, free DNA; lane 2, 200nM endonuclease over-produced by the pJS4M-pACR124 plasmid system; lane 3, as lane 2 plus 1mM HsdR; lane 4, 200nM endonuclease over-produced by the pJS4M-pBGSR124 plasmid system; lane 5, as lane 4 plus 1mM HsdR; lane 6, as lane 4 plus 200nM methylase; lane 7, 200nM methylase; lane 8, 200nM methylase plus 200nM HsdR; lane 9, 200nM methylase plus 1200nM HsdR. Positions of individual DNA-protein complexes and unbound DNA on the gel are indicated.

Figure 2 Subunit composition of the gel-retarded protein-DNA complexes of EcoR124I

The histograms compare relative ratios of subunits extracted from the two DNA-protein complexes of purified EcoR124I endonuclease produced by pJS4M-pBGSR124 plasmid system (complexes I and II shown in Figure 1) to the relative ratios of subunits in a series of mixtures of HsdR and methylase, as determined by densitometric scanning of SDS-PAGE gels stained with Coomassie Blue. White bars, HsdS; grey bars, HsdM; black bars, HsdR. Panels 1-5 on the x-axis correspond to the following HsdR:MTase ratios: 0.5:1, 1:1, 2:1, 3:1, 4:1. Panels 6 and 7 are complex I and complex II, respectively. The data suggest that the stoichiometry of the species in complex I is R₁M₂S₁, and the stoichiometry of the species in complex II is R₂M₂S₁.

Figure 3 Titration of EcoR124I methylase with HsdR, followed by measurement of DNA cleavage

A mixture of 200nM methylase and 200nM pDRM-1R DNA ( a plasmid with a single EcoR124I recognition site) was incubated with increasing concentration of HsdR in the presence of 0.2mM AdoMet and 10mM ATP. Concentration of linear DNA produced after 5 min incubation (a) was plotted against HsdR:MTase molar ratio. Reaction products were quantified as described elsewhere (Janscak et al., 1996). The lines drawn are only to guide the eye.

Figure 4 Titration of EcoR124I methylase with HsdR, followed by measurement of ATPase activity

A mixture of 200nM methylase and 200nM pDRM-1R DNA was incubated with increasing concentration of HsdR in the presence of 0.2mM AdoMet and 10mM ATP. The rate of ATP hydrolysis (b) was plotted against HsdR:MTase molar ratio. Reaction products were quantified as described elsewhere (Janscak et al., 1996). The line drawn are only to guide the eye.

Figure 5 Titration of methylase with HsdR followed by HPLC gel filtration

The molecular weight, determined by HPLC gel filtration, of the complex formed between methylase and HsdR subunit as the HsdR subunit concentration is increased. The methylase concentration was 420nM throughout. The samples were applied to the column immediately after mixing (open circles), or after 24 hours incubation (filled circles). The lines drawn are only to guide the eye.

Figure 6 Dissociation of the EcoR124I endonuclease complex

Dissociation of R₂M₂S₁ into R₁M₂S₁ and HsdR was monitored by a gel retardation assay with the 39-mer oligoduplex containing one EcoR124I recognition site. The endonuclease was reconstituted by mixing the methylase and HsdR in a molar ratio of 1:2. A series of dilutions of this mixture in a concentration range from 1000nM to 5nM was incubated with 20nM DNA at room temperature for 10 minutes and subsequently analysed on a 5% non-denaturing polyacrylamide gel (a). Lanes 1-12 correspond to the following protein concentrations: 1000, 500, 200, 150, 100, 80, 60, 40, 20, 10, 5, 0 nM. The gel was quantified using a phophoroimager and the percentage of R₂M₂S₁-DNA complex of the total bound DNA was plotted against protein concentration (b).

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