Mutational analysis of the CG recognizing DNA methyltransferase SssI: insight into enzyme-DNA interactions.
To characterize important steps of DNA methylation by M.SssI, a prokaryotic DNA-(cytosine C5)-methyltransferase (C5-MTase) sharing the specificity of eukaryotic C5-MTases (5'-CG-3'), ten amino acids, selected on the basis of sequence alignments and a computational model, were subjected to mutational analysis. Wild-type and mutant M.SssI variants were studied to determine methylation activity, DNA binding affinity, capacity to induce base flipping, and ability to form covalent complex with a DNA substrate containing the mechanism-based inhibitor 2-pyrimidinone. Wild-type M.SssI induced strong fluorescence when bound to substrate DNA containing 2-aminopurine in place of the target cytosine, indicating flipping of the target base. Reduced fluorescence, moderate, or drastic loss of methyltransferase activity and reduced DNA binding suggest the involvement of the conserved S145 (motif IV), R232 (motif VIII, QxRxR), and T313 (variable region, conserved TL), as well as of the non-conserved Q147 in base flipping. Replacement of E186 (motif VI, ENV) and R230 (motif VIII, QxRxR) with alanine resulted in loss of methyltransferase activity without impairing DNA binding affinity. These data are consistent with the catalytic role of E186 and R230, and provide, for the first time, experimental support for the essential function of the hitherto not investigated invariant arginine of motif VIII in C5-MTases.
Darii
Maria V
MV
Chemistry Department, Moscow State University, Moscow, 119991, Russia.
Cherepanova
Natalia A
NA
Subach
Oksana M
OM
Kirsanova
Olga V
OV
Raskó
Tamás
T
Slaska-Kiss
Krystyna
K
Kiss
Antal
A
Deville-Bonne
Dominique
D
Reboud-Ravaux
Michèle
M
Gromova
Elizaveta S
ES
D000595:Amino Acid Sequence; D002384:Catalysis; D019175:DNA Methylation; D004252:DNA Mutational Analysis; D015257:DNA-Cytosine Methylases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D008969:Molecular Sequence Data; D016415:Sequence Alignment
C081573:DNA modification methylase SssI; D015257:DNA-Cytosine Methylases