Probing the protein-DNA interface of the EcoRV modification methyltransferase bound to its recognition sequence, GATATC.
The DNA contacts produced between the EcoRV modification methyltransferase and its recognition sequence, GATATC, have been determined. The enzyme's general location in a methylase/DNA/sinefungin ternary complex was evaluated by protection from exonuclease III digestion. Important phosphate contacts were resolved using N-ethyl-N-nitrosourea ethylation interference footprinting. Methylation protection and interference using dimethyl sulfate were employed to assess significant contacts to purinic bases. The protein-DNA interface was further probed using oligodeoxynucleotides containing base analogues within the GATATC sequence. Most of the experiments were carried out using hemimethylated sequences, i.e., having 6-methyladenosine at the methylation site in one of the strands. The monomeric methylase was found to bind to the DNA in two different orientations for the methylation of each strand. The enzyme approaches the DNA, predominantly from one "side", and makes most of its contacts in the major groove. In either of the two binding events contacts are made to the four phosphates NpNpNpGpA and the three bases GAT (where GAT represents the 5' half of the GATATC site) on both DNA strands. The phosphates and bases in the 3' ATC half are much less important. Although the contacts made to the equivalent locations on each strand are similar, they display a slight but consistent change dependent on which strand contains the 6-methyldeoxyadenosine. This strand variation shows completely reciprocal behavior, switching around exactly, depending entirely on the methylated deoxyadenosine location. It is this that provides evidence for the two binding modes. The results obtained are discussed in terms of possible models for the protein-DNA interface.
Szczelkun
M D
MD
Department of Biochemistry, University of Southampton, U.K.
Jones
H
H
Connolly
B A
BA
D000241:Adenosine / Q000031:analogs & derivatives / Q000494:pharmacology; D001483:Base Sequence; D001665:Binding Sites; D003196:Computer Graphics; D004247:DNA / Q000737:chemistry / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology; D005038:Ethylnitrosourea / Q000378:metabolism; D005090:Exodeoxyribonucleases / Q000378:metabolism; D008745:Methylation / Q000187:drug effects; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D009838:Oligodeoxyribonucleotides / Q000737:chemistry / Q000378:metabolism*; D010710:Phosphates / Q000378:metabolism / Q000494:pharmacology; D015265:Site-Specific DNA-Methyltransferase (Adenine-Specific) / Q000737:chemistry / Q000378:metabolism*
D009838:Oligodeoxyribonucleotides; D010710:Phosphates; D004247:DNA; C056127:DNA modification methylase EcoRV; D015265:Site-Specific DNA-Methyltransferase (Adenine-Specific); D005090:Exodeoxyribonucleases; C020133:exodeoxyribonuclease III; D000241:Adenosine; D005038:Ethylnitrosourea; C006235:sinefungin