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From the mutant analysis we conclude that a) specific RNA binding is not absolutely essential for DNA demethylation and b) that G39 is a critical amino acid for the function and localization of Gadd45a.
Based on the crystal structure with Brd2 and site directed mutant analysis, we assigned this shifted resonance to Y139.
For the subsequent mutant analysis we used the procedure described in detail for the identification of the essential genes in Porphyromonas gingivalis[ 21].
From the H99 mutant analysis, we know that one or more of the H99 genes are important for GMR- Dp53-induced apoptosis.
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In the double-mutant analysis, we observed that ga1 and cpd generated late flowering, and that ga1 could enhance the cpd phenotype (Figure 2).
To be able to take into consideration these differences in our systematic in silico mutants analysis, we used the PoP of the elements after 20 millions of steps even though the simulations were not at steady-state yet.
In addition, by using double mutant cycle analysis, we probed the energetic contribution of selected pairs of amino acid residues.
Using X-ray crystallography and multiple-mutant cycle analysis we demonstrated the added complexity in understanding non-Ala mutations.
We have added mutant analysis, to complement morpholino data, wherever possible.
We show by mutant analysis that only hid, but not rpr, is required for Dp53-induced apoptosis in this system.
To demonstrate and reproduce these studies in a larger variety of embryos and to include the mutant in the analysis, we applied a different functional test.
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