Mutation Experiments
Guided by the structural analysis, certain key conserved residues were selected for mutation experiments. Firstly, mutation were introduced to the hollow concave surface in which the methyl group of the methylated cytosine is harbored. Such mutations involved :
1. Trp1512 → Ala mutant is catalytically dead, proving the importance of Trp1512 in base-stacking of methyl-cytosines.
2. Other similar mutation lead to reduction by a factor of 2 to 4 in hemi-methylated CpG substrate and 3 to 4 for non-methylated DNA. From these findings we can conclude that the clusters of residues determine substrate specificity instead of individual amino acids.
3. Met1235 → Ala mutant showed reduced activity in both hemi- and non-methylated DNA.
4. Lys1537 → Ala mutant strangely shows decrease in catalytic activity in hemi-methylated DNA but an increase in non-methylated DNA. Although there is no proof of, it is speculated that this muation causes alteration in the orientation of tryptophan residues and thus alters substrate recognition.
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| Methylation rate in mutant DNMT1[taken from http://www.sciencemag.org/content/331/6020/1036.full#F1 ] |
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