| Cells use two major mechanisms to repair double strand breaks (DSBs): homologous recombination and nonhomologous end joining (NHEJ). Nonhomologous repair mechanisms have been relatively less well studied, despite their potential importance in generating chromosomal rearrangements that lead to cancer and other abnormalities. As described in Chapter I, we have developed a novel genetic assay in Saccharomyces cerevisiae to identify and characterize chromosomal rearrangements associated with nonhomologous repair of a unique induced DSB. Using this assay system we find that about 1% of wild type haploid cells successfully repair the break, and 1% of repair events involve chromosomal rearrangements consisting of insertions, deletions, translocations or inversions.; As discussed in Chapter II, two different types of extrachromosomal DNA molecules, Ty1 cDNA intermediates and mitochondrial DNA fragments, can be captured at the break site. These events are YKU80-dependent, indicating that they are a form of nonhomologous end joining.; In Chapter III, we show that deletions, ranging from 163 to 12877 bp, can be associated with nonhomologous repair of a unique DSB. These deletions fall into two categories, depending on size, with the largest deletions being independent of YKU80. This novel form of deletion formation is characterized in detail.; More complex chromosomal rearrangement events involving exchange of broken chromosome arms were also identified. In chapter IV, we show that cells may undergo reciprocal translocations to rejoin two simultaneous breaks. The pattern of joining of these events is dependent on the genetic background. Further, we identified translocations and inversions after a single cut. These events are RAD52-independent and YKU80-dependent.; In chapter V, we define an inefficient recombination pathway revealed by our genetic assay that occurs during gene conversion from an ectopic donor locus. We have investigated the roles of the mismatch repair genes, MSH2, MSH6 and PMS1 in this process.; Using our assay, various chromosomal rearrangements have been examined which depend on the specific genetic background of the cells under study. These events occur by different mechanisms that are either competitive or cooperative. Our assay can be applied to a number of problems associated with chromosome stability and DNA repair. |
