EcoR124I MTase

The M.EcoR124I MTase

We have cloned the genes coding for the two subunits (HsdM and HsdS) of the Type-I DNA methyltransferase (MTase), M.EcoR124, into the specially constructed expression vector, pJ119 Patel (1992). These subunits have been synthesized together as an intact DNA MTase. We have also cloned the individual subunit-encoding genes under the control of the T7 gene 10 promoter or the lacUV5 promoter. High levels of expression have been obtained in all cases. While HsdM was found to be soluble, HsdS was found to be insoluble. However, in the presence of the co-produced HsdM subunit, HsdS was found in the soluble fraction as part of an active DNA MTase. We have purified the cloned multi-subunit enzyme and shown that it is capable of DNA methylation both in vivo and in vitro.

We have also developed a complementation assay which allows us to distinguish between mutations affecting subunit assembly and mutations affecting DNA binding in the DNA recognition subunit (HsdS) of the multimeric restriction endonuclease EcoR124I. A number of random point mutations were constructed to test the validity of this assay Abadjieva (1993). Two of the mutants produced were found to be truncated polypeptides that were still capable of complementation with the EcoR124I Hsd subunits to give an active restriction enzyme of novel DNA specificity. The N-terminal variable domain (responsible for recognition of GAA from the EcoR124I recognition sequence GAAnnnnnnRTCG) and the spacer region (central conserved region) is intact in both of these mutants. One of these mutant genes (hsdS(D50)) has been cloned as an active Mtase. Purification of the MTase proved to be difficult because the complex is weak. However, MTase activity was obtained from a soluble cell extract, and this allowed us to determine the DNA recognition sequence of the Mtase to be GAAnnnnnnnTTC. This recognition sequence is an inverted repeat of 5'-end of the EcoR124I recognition sequence. This suggests that the mutant MTase is assembled from two inverted HsdS(D50) subunits, possibly held together by the HsdM subunits Abadjieva (1993).

The DNA recognition subunit (HsdS) of Type I restriction endonucleases can be divided into domains by means of amino acid identity between subunits from the same family. It has been proposed that DNA-protein interactions occur within the variable domains of the subunit and that protein-protein interactions involve the conserved domains. We have constructed a number of deletion mutants of HsdS that have allowed us to investigate protein-protein interactions. Using a combination of the "competitive" complementation assay and the ability of HsdM to "solubilise" HsdS, we have defined a region within the central conserved domain of HsdS which is responsible for HsdS-HsdM interaction. Computer analysis of amino acid identity between the N-terminal half and the C-terminal half of HsdS identifies a region (repeated in both conserved domains), one copy of which overlaps the region we have identified as essential for HsdS-HsdM interactions, which may be responsible for such protein-protein interactions Abadjieva (1994).

We have recently produced a series of point mutations within this central conserved domain in an attempt to identify amino acids responsible for protein-protein interactions. Classically mutations within the hsdS gene have been Res^- Mod^- because they are affected in their DNA binding, however, when we screened two hundred point mutations (including some multiple mutants) we identified two mutants with a non-classical phenotype. The phenotype was Res^- Mod⁺, indicating the mutation cannot affect DNA binding because the mutant is still capable of methylation (this would be affected by mutation which affected DNA binding. It is tempting to suggest that this mutation is directly altering protein-protein interactions between HsdS and HsdR, and thus preventing the HsdR subunit from restricting DNA. However, this is unlikely, and at this time there is no direct evidence for protein-protein contacts between HsdS and HsdR, therefore it is more likely that this mutation affects the HsdR subunit indirectly perhaps by altering slightly the position of HsdM and thus preventing HsdR assembling correctly Weiserova (1997).

The position of this mutation is at the border of the central conserved domain and the second target recognition domain (TRD2). The altered amino acid is a tryptophan which has been changed to an arginine. Such a change from a hydrophobic residue to a positively charged residue would be expected to produce a major structural alteration.

Diagram showing the location of the non-classical mutation in hsdS

Diagram showing a series of deletion mutations of the hsdS gene which identify a region important for protein-protein interactions

Diagram showing the location of a collection of DNA-binding mutants in hsdS

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