An alternative approach to study the enzymatic specificities of the CfrBI restriction–modification system

Abstract

Restriction–modification systems (RMS) are the main gene-engineering tools and a suitable model to study the molecular mechanisms of catalysis and DNA–protein interactions. Research into the catalytic properties of these enzymes, determination of hydrolysis and DNA-methylation sites remain topical. In our previous work we have cloned and sequenced the CfrBI restriction–modification system (strain Citrobacter freundii) , which recognizes the nucleotide sequence 5′-CCWWGG-3′. In this article we describe the cloning of the methyltransferase and restriction endonuclease genes (gene encoding CfrBI DNA methyltransferase ( cfrBIM) and gene encoding CfrBI restriction endonuclease ( cfrBIR) ) separately to obtain strains overproducing the enzymes of this system. His 6 -CfrBI, which had been purified to homogeneity, was used to establish the DNA-hydrolysis point in its recognition site. CfrBI was shown to cleave DNA after just the first 5′C within the recognition site and then to generate 4-nt 3′ cohesive ends (5′-C/CWWGG-3′). To map the site of methylation by M.CfrBI, we exploited the fact that the Cfr BI site partially overlaps with the recognition sites of the well-documented enzymes KpnI and ApaI. The M.CfrBI- induced hemimethylation of the internal C residue of the ApaI recognition sequence (GGGC N4m CC) was observed to block cleavage by ApaI. In contrast, KpnI was able to digest its M.CfrBI-hemimethylated site (GGTA N4m CC). KpnI was used to restrict a fragment of DNA harbouring the CfrBI and KpnI sites, in which the CfrBI site was methylated in vitro by His 6 -M.CfrBI using [ 3 H]-SAM. The subsequent separation of hydrolysis products by electrophoresis and the enumeration of incorporated [H3]-methyl groups in each of the fragments made it possible to determine that external cytosine undergoes modification in the recognition site.