Localization
of recombination intermediates in S. cerevisiae using GFP-based
variants in vivo.
Christian Müller (1), Michael Lisby (2), Rodney Rothstein (2), Uffe H. Mortensen
(1)
(1) Center of Process Biotechnology, Biocentrum-DTU, Søltofts Plads, Building
223, Technical University of Denmark (cm@biocentrum.dtu.dk); (2) Department of
Genetics and Development, Columbia University College of Physicians and
Surgeons, New York, New York 10032-2704, USA.
The generation of a DNA double strand break (DSB) in the genome is a potentially catastrophic event that induces cell-cycle arrest and may ultimately result in cell death. In the yeast, S. cerevisiae, such lesions are predominantly repaired by homologous recombination - a process that depends on proteins encoded by the RAD52 epistasis group of genes: RAD50-59, MRE11, XRS2 and RFA1. Among these, RAD52 plays a key role and defects in this gene cause the most dramatic repair and recombination defects. A key step in homologous recombination is when the damaged DNA strand invades an intact homologous sequence. In vitro, Rad51/RP-A has been shown to catalyze a DNA strand invasion reaction alone, but addition of Rad52 greatly improves the efficiency of this reaction. However, it is unclear whether Rad52 acts on these substrates before or during strand invasion. Previously, we have developed a visual assay to detect ongoing recombination and DNA DSB repair in vivo [1]. This assay employs a functional fusion of Rad52 and GFP. We described that Rad52-GFP redistributes after DNA damage to form bright distinct foci. We intend to further develop this visual recombination assay by taking advantage of the availability of GFP variants with different spectral properties. This assay will be utilized to visualize homologous recombination in vivo. [1] Lisby et al. 2001 PNAS 98: 8159-8922.