The structural basis of DNA recognition and base extrusion by a DNA cytosine-5 methyltransferase M.HaeIII.

摘要

The goal of this study is to elucidate the mechanism of sequence specific DNA recognition and base extrusion by DNA cytosine-5 methyltransferase from Haemophilns aegyptius M.HaeIII. We have solved the crystal structure of the C71S mutant of M.HaeIII in complex with the substrate DNA at 2.4.Å resolution (InC below for brevity). For the first time an X-ray structure reveals a fully intrahelical target cytosine poised for extrusion by a cytosine-5 methyltransferase. The target cytosine is destabilized, having lost most of its stacking interactions with both neighbouring bases and making longer hydrogen bonds with the complementary guanine. In addition the protein competes for Watson-Crick hydrogen bonding of the target base pair. Both the protein and the DNA conformations are remarkably different from those in the structure where the target cytosine is extrahelical (ExC for brevity) [57]. In the ExC structure the cytosine 3' of the target base is the one forming a base pair with the guanine of the target base pair, whereas in the InC structure the bases within the recognition sequence stay correctly paired. The conformation of the DNA backbone 3' to the target cytosine changes significantly as well - it shifts further away from the protein. The catalytic loop of M.HaeIII (residues 71–89) is retracted away from the DNA as well. The results suggest that M.HaeIII actively participates in base flipping. It destabilizes the target base by altering both stacking and Watson-Crick hydrogen bonding two fundamental interactions that keep DNA bases intrahelical.;In order to elucidate the role of the intercalating residue Ile-221 in base extrusion, a glycine mutant was constructed. Its structure in complex with the specific DNA has been determined using X-ray crystallography. This structure is essentially identical to the ExC, suggesting that the extrahelical state, observed in ExC, can be achieved in the absence of the DNA helix stabilization provided by Ile-221.