Toward Quantitative Measurements of DNA Repair Protein Recruitment Dynamics in Live Cells

Authors

Matthew K. Daddysman, Christopher J. Fecko

Abstract

The three cellular processes that manipulate DNA, transcription, replication, and repair, are driven by proteins that exist in a rather low copy number. One of the open questions in biology is how do these proteins, particularly in the cases of transcription and repair, find specific binding sites that are miniscule in comparison to the size of the genetic code with enough efficiency for life to exist. In vitro space experiments with purified DNA and transcription and repair complexes have explained much about the kinetics of these process; however, the in vivo environment of DNA differs greatly. Therefore, it is necessary to design methods to probe these processes in vivo to ascertain the kinetics of these processes in a cellular environment. Our goal is to interrogate the spatiotemporal dynamics of DNA repair using live-cell microscopy. We use a laser scanning multiphoton microscope to generate well defined regions of photolesions as well as conduct fluorescence recovery after photobleaching (FRAP) experiments. Our research focuses on recruitment kinetics in the polytene cells of the Drosophila melanogaster. The large, distinct chromosomes during interphase allow us to conduct kintetic measurements of protein movement in the chromatin and in the interchromatin space. Such distinctions are not possible in normal cells where the chromatin is diffuse.