Single-cell visualization of DNA repair
The creation of a DNA double strand break constitutes the most dangerous type of DNA damage. Inefficient response to DNA damage may lead to hypersensitivity to cellular stressors, susceptibility to genomic defects and resistance to apoptosis, which can lead to cancer. Current research on DNA repair has enabled numerous breakthroughs in our understanding of the DNA repair mechanisms at the population level. However, population level measurements by nature have two fundamental shortcomings: First, they are only sensitive to the mean of a distribution and usually hide the standard deviation and the cell-to-cell variability of the repair processes. Second, the population level measurements do not visualize the repair process and therefore the exact mechanism by which the donor and recipient sequences are brought together is not well understood. Single cell visualization of DNA repair in vivo is a powerful new technique, which addresses both of these aspects. Because quantitative understanding of the DNA repair mechanism at the level of single cells has been lacking, the impact of epigenetic individuality on DNA repair has also been ignored. To proceed further it is critical to acknowledge that cells even in genetically identical population exhibit individuality, which has important implications on the fitness of a population.
My lab utilizes a multidisciplinary approach, to understand the mechanism of DNA repair during the mating type switching in yeast. By fluorescently tagging several locations of DNA (Fig. 1), we visualize the dynamics of yeast chromosome III, during and after the formation of a very well defined double-strand break. Additionally by tagging several key proteins involved in the repair process, the exact timing of the repair can be quantitatively measured. These quantitative measurements will be used to formulate a predictive mathematical model of the repair process, based on polymer models of chromosomes.
We use a microfluidic device to enable rapid exchange of different media over the cells, thereby allowing precise regulation of the cells microenvironment (in collaboration with Amy Rowat from David Weitz's lab at Harvard University) (Fig. 2). My lab is actively collaborating with the Haber Laboratory (Biology, Brandeis).
Figure 1: Chromosome III of yeast is tagged in 2 different locations
The DNA dynamics during the repair is visualized by inserting arrays of LacO binding LacI-GFP at sequences near the donor and recipient DNA.
Figure 2: The Progeny chamber
The progeny of a single cell grow in the channels. The depth of the channels and the size of the cells are comparable (5 mm) and therefore cells do not grow out of focus, which allows for long-term microscopy over many cellular generations. The cells grow with doubling time of approximately 90 minutes, which is consistent with the bulk growth rate measurement and demonstrates optimal growth conditions.