The Casellas lab is headed by a new PI interested in genomic integrity. They are combining confocal imaging, GFP fusions, and biochemistry to address the central question of how DNA damage modulates transcription. This lab plans to install ProcessDB because they are “convinced this is the way to analyze GFP experiments.”
Initial hypothesis for mechanism of DNA damage, based on blockade of elongation, was drawn using familiar symbol-and-arrow diagrams
Investigators’ knowledge of molecular abundances and relative speeds of reactions was captured in ProcessDB tables. When model parameters are unknown, ProcessDB supplied biologically reasonable values that were then tested against experimental data.
ProcessDB automatically generates all the appropriate equations necessary to quantify the investigtors’ diagram
Experimental protocols with FRAPs of individual nucleoli at various times after DNA damage were entered into ProcessDB Experiment forms.
ProcessDB merged multiple experimental protocols with the quantitative model and exported the combined ‘Model of Experiments’ to the Berkeley Madonna solver for simulation and optimization
Initial hypothesis failed to account for the overshoot seen in FRAP of RPA194 10 seconds after DNA damage
Seven modifications of this hypothesis were readily constructed using the ProcessDB database and diagram tools.
Again, the hypotheses were rejected, but the simulations suggested an alternative hypothesis in which the effects of damage are mediated through promoter sequestration.
The promoter sequestration hypothesis was easily constructed using earlier models as a foundation (see diagram)
Exporting this new hypothesis combined with the control and post-damage FRAP protocols, followed by parameter optimization showed the promoter sequestration model is quantitatively consistent with the experimental data (see graphs) thus making a strong case for a novel hypothesis