PARENT SESSION
1:30 PM to 3:30 PM
Monday, April 22, 2002
Poster Session 15 DNA Damage and Repair II
Room: Nevada Exhibition Center
(P20-202) Strict regulation of recombination in the rDNA array.
Rutherford, Erin*,1, Max, Tamara1, Nickoloff, Jac2, Ruscetti, Tracy1, 1 Bioscience Division, Los Alamos, NM2 Department of Molecular Genetics and Microbiology, Albuquerque, NM
ABSTRACT-
In the budding yeast, Saccharomyces cerevisae , double strand breaks are most often repaired by homologous recombination (HR). In tandem repeats, such as the ribosomal DNA (rDNA) gene array, repair via HR with associated crossover could result in the loss of repeats and may be detrimental. We have developed a rDNA-specific recombination reporter system based on two non-functional ura3 alleles flanking LEU2. In one allele (the recipient allele), a specific double strand break can be introduced by induced expression of HO endonuclease. The second allele (the donor allele) is used to template repair. We compared repair of DSBs both inside and outside the array (at the Ura3 locus). We found that the frequency of recombination was lower inside the rDNA and that crossover events (that may lead to circle formation) were extremely rare. We also found that inside the rDNA array, recombination tracts were not as long as repair tracts outside the rDNA. Furthermore, we determined that inside the rDNA, the tracts were extremely bias to extend in only one direction. These results suggest that rDNA recombination is strictly regulated. To investigate the regulatory mechanism of rDNA recombination, we examined the effects of Sir2, a protein responsible for gene silencing of rDNA and a good candidate for recombination regulation. We found that mutation in sir2 increased the frequency of recombination in rDNA and increased tract length. Mutation of sir2 had no effect on tract directionality or crossover frequency. Another protein that appears to control rDNA specific recombination is the replication fork block protein, Fob1. By mutating FOB1 and analyzing rDNA specific recombination, we will be able to 1) determine the extent to which FOB1 controls rDNA recombination, and 2) determine how replication and recombination processes are linked in the rDNA.
KEYWORDS: Recombination, Ribosomal DNA, DNA repair, Saccharomyces cerevisae