Expression, purification, characterization of DNA binding activity and crystallization of a putative type II DNA Cytosine-5-methyltransferase from Microcystis aeruginosa

Abstract

DNA 5-methylcytosine modification plays an important role in the regulation of a variety of biological functions in both prokaryotic and eukaryotic organisms. Previous studies show that DNA Cytosine-5-methylation is predominantly associated with restriction-modification system in bacteria. IPF4390 is deduced to be a putative type II DNA Cytosine-5 methyltransferase from a fresh water cyanobacterium, Microcystis aeruginosa. Both its substrate sequence specificity and catalytic mechanism need to be revealed. In this study, the cloning, expression, purification, DNA binding assays and crystallization of IPF4390 are reported. Results of DNA binding assays demonstrate that IPF4390 can specifically recognize and bind two double-stranded DNAs containing GGNCC (N = A, T, C or G) sequences (HgiBI: 5'-ATAAGGACCAATA-3'; TdeIII: 5'-ATAAGGGCCAATA-3'). Therefore, IPF4390 is probably capable of blocking endonuclease cleavage once restriction sites containing these sequences. Moreover, the crystal of IPF4390 in the presence of TdeIII was obtained, and its X-ray diffraction data were collected and scaled to a maximum resolution of 2.46 Å.

Keywords: Crystallization; DNA Cytosine-5-methyltransferase; DNA binding; Microcystis aeruginosa; Prokaryotic expression; Protein purification.