DOE Low Dose Radiation Research Program Workshop I Abstracts November 10-12, 1999, Washington, D.C.

20. The Dynamic Behavior of Broken Chromosomes in Yeast

Kerry Bloom, Douglas A. Thrower, L. Kevin Lewis, and Michael A. Resnick
Department of Biology, CB3280, University of North Carolina, Chapel Hill, NC 27514
kbloom@email.unc.edu

Summary: We will observe the movements of both ends of a broken chromosome in yeast to determine the functions of previously identified DNA repair genes.

Abstract: Two major DNA double-strand break (DSB) repair pathways, homologous recombination and nonhomologous end joining, have been characterized in both the budding yeast Saccharomyces cerevisiae and in humans. A significant number of proteins have been identified whose functions within these pathways are necessary for the repair of DNA double strand breaks. The requirement for each of these proteins at specific steps of the DNA repair process has yet to be determined. We propose to use yeast as a model system to study the dynamics of chromosomes as they undergo breakage and repair. Initial studies will examine the cytological fate of both ends of a single chromosomal DSB induced by HO endonuclease using video imaging of fluorescently tagged chromosomal DNA in living cells. The fate(s) of both ends will be followed in haploid and diploid cells in both G1 and G2 to assess the impact of increased or decreased opportunities for DSB repair by homologous recombination. Real time analysis of the metabolism of a broken chromosome will be performed in mutant strains that are defective in both major pathways of DSB repair as well as for strains containing a deletion of the checkpoint gene RAD9. Our progress thus far includes the successful construction of a chromosome marker cassette that can be integrated into the yeast genome at any desired location and confirmation that movements of a chromosome containing this marker can be easily followed by microscopic imaging system. We have currently integrated this marker at either side of HO cut sites located on two different chromosomes. We are also in the process of developing a system utilizing different spectral variants of green fluorescent protein, allowing multiple structures or multiple chromosome markers to be easily imaged in the same cell. We have also examined the effect of a deletion of the yeast homologue of the nonhomologous end joining protein Ku70 on repair of dicentric chromosome breakage and have found that a loss of Ku results in a loss of chromatin structure between the dicentric centromeres.

Given the number of yeast DNA repair genes that have human structural homologues we anticipate our findings in this project will identify specific roles of genes necessary for the repair of DNA damage that results from exposure to low dose radiation.