Functional studies of the small subunit of EcoHK31I DNA methyltransferase.
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 alpha and beta. Polypeptide beta 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 alpha/beta complex recognizes specific oligonucleotide substrates. Polypeptide alpha formed aggregates with DNA, while polypeptide beta alone did not bind DNA. Therefore, polypeptide beta assists in the proper binding of polypeptide alpha to DNA substrate. The complex of polypeptide alpha and a polypeptide beta 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 (Kd) of the alpha/beta complex was 56.4 nM, while the Kd value for the alpha/deltaN46-polypeptide beta complex was increased approximately 95-fold, caused by a drastic decrease in dissociate rate constant (kd) and an increase in the association rate constant (ka). This indicates that the N-terminal region of polypeptide beta takes part in subunit interaction, while the C-terminal region is involved in DNA binding.
Fung
Wai-To
WT
Department of Biochemistry and Centre for Protein Science and Crystallography, The Chinese University of Hong Kong, Shatin, NT, Hong Kong, China.
Sze
Kong-Hung
KH
Lee
Kai-Fai
KF
Shaw
Pang-Chui
PC
D001483:Base Sequence; D002942:Circular Dichroism; D015257:DNA-Cytosine Methylases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D024202:Electrophoretic Mobility Shift Assay; D004926:Escherichia coli / Q000201:enzymology*; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D020836:Protein Structure, Quaternary; D017433:Protein Structure, Secondary; D021122:Protein Subunits / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000378:metabolism; D017384:Sequence Deletion / Q000235:genetics; D020349:Surface Plasmon Resonance
D029968:Escherichia coli Proteins; D021122:Protein Subunits; D011994:Recombinant Proteins; C092246:DNA modification methylase EcoHK31I; D015257:DNA-Cytosine Methylases