| Abstract [eng] |
The ability of proteins to discriminate between modified and unmodified cytosine is of key importance for such processes as epigenetic regulation in eukaryotes and activity of methyl-directed restriction endonucleases (REases) in bacteria. In eukaryotes modified cytosine is recognized either in the context of dsDNA (by the MBD domains), or in the flipped-out state (by the SRA domains). Structurally related SRA-like domains are also found in methyl-directed REases of MspJI and PvuRts1I families. Another methyl-directed endonuclease McrBC, despite an unrelated structure, also employs the base flipping mechanism. However, the mechanism of modified cytosine recognition by many uncharacterized eukaryotic and prokaryotic modified cytosine “readers” remains unknown, as is the DNA recognition and reaction mechanism of the classical methyl-directed enzyme McrA. In this study using variuos chemical methods we demonstrate that MspJI and PvuRts1I enzymes flip out modified base from dsDNA. We also identify protein loops in MspJI family enzymes that act as DNA binding/recognition modules, replacement of which results in altered sequence specificity, and present the first detailed study of McrBC specificity for various cytosine modifications. Finally, this is the first study that demonstrates McrA nuclease activity in vitro and confirms functions of its N-terminal (DNA binding) and C-terminal (catalysis) domains. Our findings pave the way for engineering of new tools for epigenome studies. |
