Binding of MmeI restriction-modification enzyme to its specific recognition sequence is stimulated by S-adenosyl-L-methionine.
Restriction endonucleases serve as a very good model for studying specific protein-DNA interaction. MmeI is a very interesting restriction endonuclease, but although it is useful in Serial Analysis of Gene Expression, still very little is known about the mechanism of its interaction with DNA. MmeI is a unique enzyme as besides cleaving DNA it also methylates specific sequence. For endonucleolytic activity MmeI requires Mg(II) and S-adenosyl-l-methionine (AdoMet). AdoMet is a methyl donor in the methylation reaction, but its requirement for DNA cleavage remains unclear. In the present article we investigated MmeI interaction with DNA with the use of numerous methods. Our electrophoretic mobility shift assay revealed formation of two types of specific protein-DNA complexes. We speculate that faster migrating complex consists of one protein molecule and one DNA fragment whereas, slower migrating complex, which appears in the presence of AdoMet, may be a dimer or multimer form of MmeI interacting with specific DNA. Additionally, using spectrophotometric measurements we showed that in the presence of AdoMet, MmeI protein undergoes conformational changes. We think that such change in the enzyme structure, upon addition of AdoMet, may enhance its specific binding to DNA. In the absence of AdoMet MmeI binds DNA to the much lower extent.
Nakonieczna
Joanna
J
Intercollegiate Faculty of Biotechnology, Department of Biotechnology, University of Gdansk and Medical University of Gdansk, Kladki 24, Gdansk, 80-822, Poland. strzala@biotech.ug.gda.pl
Zmijewski
Jaroslaw W
JW
Banecki
Bogdan
B
Podhajska
Anna J
AJ
D001483:Base Sequence; D004247:DNA / Q000378:metabolism; D015252:Deoxyribonucleases, Type II Site-Specific / Q000378:metabolism*; D011485:Protein Binding; D012436:S-Adenosylmethionine / Q000378:metabolism*; D013053:Spectrophotometry; D013379:Substrate Specificity
D012436:S-Adenosylmethionine; D004247:DNA; C049760:endodeoxyribonuclease MmeI; D015252:Deoxyribonucleases, Type II Site-Specific