Differential binding of Escherichia coli McrA protein to DNA sequences that contain the dinucleotide m5CpG

Journal Article · Thu Apr 01 04:00:00 EDT 2010 · Nucleic Acids Research

DOI:https://doi.org/10.1093/nar/gkp1120· OSTI ID:1014286

Mulligan, E A; Dunn, J; Hatchwell, E; McCorkle, S R

The Escherichia coli McrA protein, a putative C{sup 5}-methylcytosine/C{sup 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.

• Research Organization: BROOKHAVEN NATIONAL LABORATORY (BNL)
• Sponsoring Organization: LABORATORY-DIRECTED RESEARCH AND DEVELOPMENT
• DOE Contract Number: AC02-98CH10886
• OSTI ID: 1014286
• Report Number(s): BNL--90687-2010-JA; YN0100000
• Journal Information: Nucleic Acids Research, Journal Name: Nucleic Acids Research Journal Issue: 6 Vol. 38; ISSN 0305-1048; ISSN NARHAD
• Country of Publication: United States
• Language: English

Related Subjects

59 BASIC BIOLOGICAL SCIENCES
99 GENERAL AND MISCELLANEOUS
AFFINITY
DNA
E. coli McrA protein
ESCHERICHIA COLI
ESMA analysis
IN VITRO
OLIGONUCLEOTIDES
PROTEINS
PURIFICATION
RESIDUES
SPECIFICITY
STRUCTURAL CHEMICAL ANALYSIS
hemimethylated DNA
pull-down assays