AI Article Synopsis
- EcoP15I DNA methyltransferase (M.EcoP15I) methylates a specific adenine in the asymmetric sequence 5'-CAGCAG-3' to protect host DNA from cleavage by the restriction enzyme R.EcoP15I.
- The enzyme is known to function as a dimer, but the exact role of this dimerization in the methylation process remains unclear.
- Research into a monomeric form of M.EcoP15I reveals that while it can bind to single-stranded DNA, it cannot perform methylation, indicating that dimerization is crucial for effective DNA binding and catalysis.
Article 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.
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| http://dx.doi.org/10.1016/j.biochi.2016.07.006 | DOI Listing |
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