Differential binding of Escherichia coli McrA protein to DNA sequences that contain the dinucleotide m5CpG.
The Escherichia coli McrA protein, a putative C(5)-methylcytosine/C(5)-hydroxyl methylcytosine-specific nuclease, binds DNA with symmetrically methylated HpaII sequences (Cm5CGG), but its precise recognition sequence remains undefined. To determine McrA's binding specificity, we cloned and expressed recombinant McrA with a C-terminal StrepII tag (rMcrA-S) to facilitate 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 N(Y > R) m5CGR is the canonical binding site for rMcrA-S. In addition to binding HpaII-methylated double-stranded DNA, rMcrA-S binds DNA containing a single, hemimethylated HpaII site; however, it does not bind if A, C, T or U is placed across from the m5C residue, but does if I is opposite the m5C. These results provide the first systematic analysis of McrA's in vitro binding specificity.
Mulligan
Elizabeth A
EA
Department of Molecular Genetics and Microbiology, Genomics Core Facility, Stony Brook University, Stony Brook, NY, USA.
Hatchwell
Eli
E
McCorkle
Sean R
SR
Dunn
John J
JJ
D044503:5-Methylcytosine / Q000032:analysis; D001483:Base Sequence; D001665:Binding Sites; D018899:CpG Islands; D004247:DNA / Q000737:chemistry / Q000378:metabolism; D019175:DNA Methylation; D004262:DNA Restriction Enzymes / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000378:metabolism*; D006801:Humans
D029968:Escherichia coli Proteins; D044503:5-Methylcytosine; D004247:DNA; D004262:DNA Restriction Enzymes; C060629:endodeoxyribonuclease McrA