of Work: The repair of DNA double-strand breaks (DSBs) and stabilization of telomeric sequences at chromosome ends involves homologous recombination and nonhomologous end-joining (NHEJ) genes in yeast. RAD50, MRE11 and XRS2 encode subunits of a nuclease complex functioning in both pathways. Defects in the related complex in humans results in Nijmegen syndrome result in increased radiation sensitivity. Employing a novel in vivo site-directed mutagenesis scheme developed in this lab, the ATP binding function of Rad50 was found to be required for both recombination and NHEJ while the nuclease function of Mre11 was required only for recombination. This structure-function analysis provides a better understanding of the process of DSB repair. The specificity of nucleases to recombination, and not NHEJ, is consistent with the finding that overexpression of Exo1 can compensate for a defect in the Rad50/Mre11/Xrs2 complex via recombination. The investigations of DSBs and repair have been extended to determining if the nature of ends determine repair capabilities and the biological consequences. Using in vivo expressed enzymes and comparing with radiation induced DSBs, it is clear that breaks with complementary ends are repaired efficiently by NHEJ while radiation induced breaks are repaired by recombination. Surprisingly, blunt end breaks are highly toxic, suggesting that agents producing these types of breaks may be the most destabilizing to genomes. In association with this analysis, a novel approach was developed for controlled, low level expression of restriction enzymes. To study DSBs directly, a new approach was developed for the in vivo characterization of a specific chromosome DSB that permit monitoring the fate of a broken yeast chromosome. The strains contain fusions of green fluorescent protein (GFP) to the E. coli LacI repressor that can bind to an integrated binding sequence array near the site of the break. A DSB resulted in rapid movements of the nucleus and """"""""breathing"""""""" between sister chromatids at the break site.
Showing the most recent 10 out of 11 publications
