DNA double‐strand break repair deficiency is associated with changes in cell cycling and cell morphology in Saccharomyces cerevisiae

Chromosomal DNA experiences a variety of different types of damage often and naturally. Considered potentially the most lethal of DNA lesions, double‐strand breaks (DSBs) involve a complete severance of both strands of DNA at some point in the chromosome. If not repaired, DSBs can lead to an increase in DNA mutations, chromosome loss events and ultimately loss in cell viability. In eukaryotes, DNA DSBs are repaired almost exclusively by two mechanisms, nonhomologous end‐joining (NHEJ) and homologous recombination (HR). NHEJ is the simpler of the two pathways, involving the direct rejoining of broken chromosome ends, while HR involves the use of a region of a homologous chromosome as a template for repair. RAD52 group proteins (Rad51, Rad52, Rad54, etc.) play an important role in the HR pathway. In Saccharomyces cerevisiae homologous recombination is considered the primary pathway for repair. Checkpoint proteins also play an important role in DSB repair by making sure that damage is repaired before a cell completes the cell cycle. If there is significant damage that requires attention, such as DSBs induced by chemicals or radiation, growth arrest is induced and the cells accumulate in G 2 phase. Yeast rad52 mutants cannot repair DSBs by HR and have elevated levels of unrepaired DNA lesions. In the current project, we have observed that rad52 cell cultures have high levels of enlarged large‐budded cells, and that log phase cells spend 64 minutes of each 128 minute cell cycle in G 2 phase. Similar results were seen with rad51 , rad54 , rad55 , rad57 , and rad59 mutants, but not rdh54 cells. Inactivation of 7 critical DNA damage checkpoint genes ( RAD9 , RAD17 , RAD24 , CHK1 , DDC1 , MRC1 , and MEC3 ) in rad52 cells, creating double mutants, abolished the high large‐budded cell phenotype. It is likely that rad52 cells have elevated levels of unrepaired DNA lesions leading to constitutively activated damage‐responsive cell cycle checkpoints. Additional experiments involving analysis of changes in nuclear and cellular morphology will be presented.