Analysis of DNA Recombination and Repair Proteins in Living Cells by Photobleaching Microscopy

Abstract

DNA double strand break repair through homologous recombination has been shown biochemically to require the coordinated action of the RAD52 group of proteins, including the DNA strand exchange protein Rad51. We have started to develop experimental tools to investigate the close cooperation of homologous recombination proteins in living cells, where proteins operate in the context of chromatin and in the presence of other nuclear processes. This chapter describes in detail methods to establish cell lines stably expressing green fluorescent protein -tagged recombination proteins and photobleaching techniques to investigate the behavior of the proteins with the use of live cell video microscopy. Fluorescence recovery after photobleaching (FRAP), fluorescence loss after photobleaching (FLIP), and their combination in the same cell are useful techniques to gain insights into the dynamic behavior of the recombination proteins. Parameters such as diffusion rates and mobile versus immobile fractions before and after DNA damage induction can be obtained. In addition, residence times of recombination proteins at sites of DNA damage can be determined. Through the application of FRAP and FLIP it is possible to establish whether proteins are present in the same multiprotein complex, whether this is affected by DNA damage induction, and whether proteins dynamically associate with and dissociate from sites of DNA damage.