title: Asymmetric DNA methylation by dimeric EcoP15I DNA methyltransferase.
abstract: EcoP15I DNA methyltransferase (M.EcoP15I) recognizes short asymmetric sequence, 5'-CAGCAG-3', and methylates the second adenine only on one strand of the double-stranded DNA (dsDNA). In vivo, this methylation is sufficient to protect the host DNA from cleavage by the cognate restriction endonuclease, R.EcoP15I, because of the stringent cleavage specificity requirements. Biochemical and structural characterization support the notion that purified M.EcoP15I exists and functions as dimer. However, the exact role of dimerization in M.EcoP15I reaction mechanism remains elusive. Here we engineered M.EcoP15I to a stable monomeric form and studied the role of dimerization in enzyme catalyzed methylation reaction. While the monomeric form binds single-stranded DNA (ssDNA) containing the recognition sequence it is unable to methylate it. Further we show that, while the monomeric form has AdoMet binding and Mg(2+) binding motifs intact, optimal dsDNA binding required for methylation is dependent on dimerization. Together, our biochemical data supports a unique subunit organization for M.EcoP15I to catalyze the methylation reaction.
authors.affiliation: Department of Biochemistry, Indian Institute of Science, Bangalore 560012, India.
authors.forename: Madhusoodanan
authors.initials: M
authors.lastname: Urulangodi
authors.affiliation: Department of Biochemistry, Indian Institute of Science, Bangalore 560012, India.
authors.forename: Rajkumar
authors.initials: R
authors.lastname: Dhanaraju
authors.affiliation: National Institute of Immunology, New Delhi 110 067, India.
authors.forename: Kanchan
authors.initials: K
authors.lastname: Gupta
authors.affiliation: National Institute of Immunology, New Delhi 110 067, India.
authors.forename: Rajendra P
authors.initials: RP
authors.lastname: Roy
authors.affiliation: Laboratory of Bioinformatics and Protein Engineering, International Institute of Molecular and Cell Biology, Trojdena 4, PL-02-109 Warsaw, Poland.
authors.forename: Janusz M
authors.initials: JM
authors.lastname: Bujnicki
authors.affiliation: Department of Biochemistry, Indian Institute of Science, Bangalore 560012, India. Electronic address: dnrao@biochem.iisc.ernet.in.
authors.forename: Desirazu N
authors.initials: DN
authors.lastname: Rao
mesh_terms: D001483:Base Sequence; D055162:Biocatalysis; D002942:Circular Dichroism; D004247:DNA / Q000235:genetics* / Q000378:metabolism; D019175:DNA Methylation; D004277:DNA, Single-Stranded / Q000235:genetics / Q000378:metabolism; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics; D009154:Mutation; D011485:Protein Binding; D017510:Protein Folding; D055503:Protein Multimerization; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism*; D012436:S-Adenosylmethionine / Q000378:metabolism; D015265:Site-Specific DNA-Methyltransferase (Adenine-Specific) / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D013050:Spectrometry, Fluorescence; D013379:Substrate Specificity
chemical_list: D004277:DNA, Single-Stranded; D011994:Recombinant Proteins; D012436:S-Adenosylmethionine; D004247:DNA; C089712:DNA modification methylase EcoP15I; D015265:Site-Specific DNA-Methyltransferase (Adenine-Specific)
keywords: DNA MTases; DNA-protein interactions; N(6)-adenine MTases; Oligomeric MTases; Protein structure-function; Type III R-M systems