The BsaHI restriction-modification system: Cloning, sequencing and analysis of conserved motifs

Robert K Neely,Richard J Roberts

doi:10.1186/1471-2199-9-48

Abstract

BackgroundRestriction and modification enzymes typically recognise short DNA sequences of between two and eight bases in length. Understanding the mechanism of this recognition represents a significant challenge that we begin to address for the BsaHI restriction-modification system, which recognises the six base sequence GRCGYC.ResultsThe DNA sequences of the genes for the BsaHI methyltransferase, bsaHIM, and restriction endonuclease, bsaHIR, have been determined (GenBank accession #EU386360), cloned and expressed in E. coli. Both the restriction endonuclease and methyltransferase enzymes share significant similarity with a group of 6 other enzymes comprising the restriction-modification systems HgiDI and HgiGI and the putative HindVP, NlaCORFDP, NpuORFC228P and SplZORFNP restriction-modification systems. A sequence alignment of these homologues shows that their amino acid sequences are largely conserved and highlights several motifs of interest. We target one such conserved motif, reading SPERRFD, at the C-terminal end of the bsaHIR gene. A mutational analysis of these amino acids indicates that the motif is crucial for enzymatic activity. Sequence alignment of the methyltransferase gene reveals a short motif within the target recognition domain that is conserved among enzymes recognising the same sequences. Thus, this motif may be used as a diagnostic tool to define the recognition sequences of the cytosine C5 methyltransferases.ConclusionWe have cloned and sequenced the BsaHI restriction and modification enzymes. We have identified a region of the R. BsaHI enzyme that is crucial for its activity. Analysis of the amino acid sequence of the BsaHI methyltransferase enzyme led us to propose two new motifs that can be used in the diagnosis of the recognition sequence of the cytosine C5-methyltransferases.

Highlights

Restriction and modification enzymes typically recognise short DNA sequences of between two and eight bases in length
Systems [2], in comparison to unique recognition sequences, there is remarkably little sequence similarity amongst the restriction enzymes and, though to a lesser extent, between the target recognition domains of the methyltransferases implying a diverse ensemble of DNA recognition modes and methods is used by these enzymes
The amino acid sequence of the restriction enzyme is in complete agreement with the previously determined N-terminal sequence of this enzyme

Summary

Introduction

Restriction and modification enzymes typically recognise short DNA sequences of between two and eight bases in length. We report the cloning, sequencing and subsequent expression and purification of the BsaHI R-M system from Bacillus stearothermophilus These enzymes target the degenerate sequence GRCGYC, where R= G/A and Y = T/C The inherent degeneracy of the DNA recognition by these enzymes provides an opportunity to study directly the mechanism of specific DNA recognition and to examine the question of how this breaks down into degenerate DNA recognition. Such enzymes are exciting targets in the ongoing effort to manipulate the recognition sequences of enzymes, for the restriction enzymes [3]

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Results

Conclusion