HhaI DNA methyltransferase uses the protruding Gln237 for active flipping of its target cytosine.
Access to a nucleotide by its rotation out of the DNA helix (base flipping) is used by numerous DNA modification and repair enzymes. Despite extensive studies of the paradigm HhaI methyltransferase, initial events leading to base flipping remained elusive. Here we demonstrate that the replacement of the target C:G pair with the 2-aminopurine:T pair in the DNA or shortening of the side chain of Gln237 in the protein severely perturb base flipping, but retain specific DNA binding. Kinetic analyses and molecular modeling suggest that a steric interaction between the protruding side chain of Gln237 and the target cytosine in B-DNA reduces the energy barrier for flipping by 3 kcal/mol. Subsequent stabilization of an open state by further 4 kcal/mol is achieved through specific hydrogen bonding of the side chain to the orphan guanine. Gln237 thus plays a key role in actively opening the target C:G pair by a "push-and-bind" mechanism.
Laboratory of Biological DNA Modification, Institute of Biotechnology, LT-02241 Vilnius, Lithuania.
Dalia
D
Daujotyte
Saulius
S
Serva
Giedrius
G
Vilkaitis
Egle
E
Merkiene
Ceslovas
C
Venclovas
Saulius
S
Klimasauskas
D015075:2-Aminopurine / Q000737:chemistry; D020029:Base Pairing; D002384:Catalysis; D003596:Cytosine / Q000737:chemistry*; D004247:DNA / Q000737:chemistry*; D015257:DNA-Cytosine Methylases / Q000737:chemistry* / Q000378:metabolism; D003903:Deuterium / Q000737:chemistry; D004305:Dose-Response Relationship, Drug; D005973:Glutamine / Q000737:chemistry*; D006859:Hydrogen / Q000737:chemistry; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D008956:Models, Chemical; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D011485:Protein Binding; D011487:Protein Conformation; D012984:Software; D013050:Spectrometry, Fluorescence; D013696:Temperature; D013997:Time Factors
D005973:Glutamine; D015075:2-Aminopurine; D006859:Hydrogen; D003596:Cytosine; D004247:DNA; D003903:Deuterium; C037664:DNA modification methylase HhaI; D015257:DNA-Cytosine Methylases