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. His6-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 CfrBI 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 (GGGCN4mCC) was observed to block cleavage by ApaI. In contrast, KpnI was able to digest its M.CfrBI-hemimethylated site (GGTAN4mCC). 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 His6-M.CfrBI using [3H]-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.