Dynamic aspects of DNA methylation in M.HhaI

• Main Author: Estabrook, R August
• Format: Dissertation
• Language: English
• Published: ProQuest Dissertations & Theses 01-01-2007
• Subjects: Biochemistry
• ISBN: 9781109911145; 1109911149

DNA methylation is ubiquitous and essential to life. In order to further our understanding of DNA methylation, facilitate drug discovery, and to elucidate unknown aspects of biology and life, we have investigated M.HhaI, a bacterial DNA cytosine methyltransferase. As M.HhaI methylates the C5 position of the internal cytosine of the recognition sequence 5'-GCGC-3', essential dynamic processes occur including base flipping, reorganization of the catalytic loop into the closed conformer, active site compression, and domain motion. By examining these processes theoretically with molecular dynamics and statistics, and experimentally with enzyme mutagenesis, kinetics, and fluorescence, we have been able to elucidate many of the underlying mechanisms which enable these reactions. The first of four studies conducted on M.HhaI involves a set of mutants that were made, in particular V121A. Amazingly, V121A had > 105-fold decrease in kcat and KDDNA which could be recovered using an abasic target base. These results supported an induced fit mechanism for DNA binding and led to the identification of a base flipping motif. The second study involved a collaborative study combining Molecular Dynamics (MD) with Statistical Coupling Analysis (SCA). This novel methodology identified amino acid pairs which are involved in correlated and anti-correlated motions responsible for catalysis. The third and fourth studies examined a specific catalytic loop of M.HhaI (residues 80-100) that undergoes a conformational rearrangement when the enzyme binds cognate DNA. To observe this rearrangement in solution, novel tryptophan residues were inserted. By monitoring fluorescence changes we show loop motion and base flipping are coupled, DNA binding is through an induced-fit, allostery from the recognition domain can control loop closure, and DNA specificity is achieved through single hydrogen bonds. In summary, these results have direct relevance for understanding M.HhaI catalysis and DNA methylation. Our conclusions also have broad relevance for understanding the processes of base flipping, the magnitude of specificity achieved by almost all DNA modifying enzymes, and the role of conformational rearrangements on enzyme catalysis. The high homology between DNA methyltransferases and their validation as clinically relevant targets for anti-cancer and antibiotic drug design makes these studies broadly applicable.