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Wai-To Fung, Kong-Hung Sze, Kai-Fai Lee, Pang-Chui Shaw

Functional studies of the small subunit of EcoHK31I DNA methyltransferase

Keywords: EMSA, Escherichia coli, methyltransferase, protein-DNA interaction, restriction-modification system

EcoHK31I DNA methyltransferase recognizes the sequence 5?-YGGCCR-3? and adds a methyl group to the fifth position of the internal cytosine to protect the DNA from cleavage by its cognate endonuclease. M.EcoHK31I is composed of polypeptides ? and ?. Polypeptide ? only contains the conserved IX motif of the C5-MTase family, and provides a unique example to show that this motif alone may be dislocated to another polypeptide. By electromobility shift assay, we found that the ?/? complex recognizes specific oligonucleotide substrates. Polypeptide ? formed aggregates with DNA, while polypeptide ? alone did not bind DNA. Therefore, polypeptide ? assists in the proper binding of polypeptide ? to DNA substrate. The complex of polypeptide ? and a polypeptide ? variant with an N-terminal deletion of 41 amino acids showed a 16-fold reduction in methylation activity. Further deletion resulted in an inactive methyltransferase. The dissociation equilibrium constant (K_d) of the ?/? complex was 56.4 nM, while the K_d value for the ?/?N46-polypeptide ? complex was increased approximately 95-fold, caused by a drastic decrease in dissociate rate constant (k_d) and an increase in the association rate constant (k_a). This indicates that the N-terminal region of polypeptide ? takes part in subunit interaction, while the C-terminal region is involved in DNA binding.

Biological Chemistry, Walter de Gruyter

Print ISSN: 1431-6730
Volume: 387, 05/2006
Pages: 507 - 513

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