Escherichia coli McrA: Construction of recombinant forms of McrA and study of its binding to methylated DNA for use as a tool in epigenetic studies

Epigenetic research has become increasingly important to the understanding of cancer. Changes in the methylation status of cytosines within the CpG islands of tumor suppressor genes and oncogenes have been linked to human cancers. Methods for determining the methylation status of individual cytosines within the CpG islands of genes affecting oncogenesis are becoming an important area of study. Current techniques often involve the use of sodium bisulfate to differentiate between methylated and unmethylted cytosine, however this technique is not practical for genome-wide analysis of cytosine methylation. The Escherichia coli McrA protein is a putative m5C-specific nuclease known to bind HpaII methylated DNA sequences (Cm5CGG). It is a potential tool for epigenetic studies determining the methylation status of CpG islands, but its precise recognition sequence has remained undefined. Our research focused on developing recombinant forms of McrA able to enrich methylated DNA fragments in affinity purification. We cloned and characterized a recombinant McrA (rMcrA) protein, which is predicted to contain a Cys 4-Zn++ finger and a catalytically important histidine triad in its putative nuclease domain. These features allow rMcrA to bind to several metal chelate resins without addition of a poly-histidine affinity tag. This observation was used to develop an efficient protocol for the rapid purification of nearly homogeneous rMcrA. The native protein is a dimer with a high a-helical content as measured by circular dichroism analysis. To aid in determining McrA's binding specificity we cloned and expressed recombinant McrA with a C-terminal StrepII tag (rMcrA-S) to aid in protein purification and affinity capture of human DNA fragments with m5C residues. Sequence analysis of a subset of these fragments, and electrophoretic mobility shift assays with model methylated and unmethylated oligonucleotides suggest that the canonical binding site for rMcrA-S is N(Y>R) m5CGR. In addition to binding symmetrically HpaII methylated double-stranded DNA, rMcrA-S binds DNA containing a single, hemimethylated HpaII site; however, it does not bind mismatches of A, C, T, or U opposite the m5C residue but does bind if I is opposite the m5C. These observations may lead to development of rMcrA-S-based m5C detection assays independent of bisulfite modification.