Vaziri

The overall objective of the proposed study is to determine how replication of the genome is limited to once per cell cycle, a key requirement for the maintenance of genomic stability. The roles of a signaling pathway involving the Rad9-Rad1-Hus1(9-1-1) checkpoint complex, in preventing DNA re-replication will be assessed. The proposal also seeks to address whether loss of re-replication control promotes gene amplification, a process commonly associated with tumorigenesis. The portion of the grant proposal that comprises the sub-contacted work centers on the role of the Husl cell cycle checkpoint protein in preventing DNA re-replication. Preliminary studies indicate that attempts at re-replication activate the 9-1-1 complex and further suggest that inhibition of 9-1-1 causes DNA re-replication. Unique reagents developed by the sub-contractor will allow direct testing of the role of the 9-1-1 complex in suppressing genome re-replication. Specifically, DNA re-replication. will be tested in murine cell lines in which the Husl gene has been constitutively inactivated by gene targeting. Because of growth defects associated with loss of Hus1, the cells additionally lack either p2l or p53, loss of either of which relieves the growth arrest associated with Husl deficiency. DNA re-replication will also be assessed in a system based on conditional inactivation of Husl in primary murine cells. This approach centers on a conditional Hus1 allele in which key coding sequences are flanked by loxP sites. These sequences can be recognized by the Cre enzyme, which promotes recombination between loxP sites and deletes the intervening sequences. By delivering Cre, via a viral vector for instance, Husl can be rapidly inactivated and the effects of Husl loss on re-replication control can be assessed in primary cells that are wild-type for p2l and p53. If warranted, future studies would address the role of Hus1 in controlling gene amplification in cultured cells and preventing ancuploidy and polyploidy in vivo, in tissues from conditional Hus1 knockout mice. The powerful genetic tools to be employed in these studies promise to elucidate the role of Hus1 in suppressing DNA re-replication and yield insights into how the 9-1-1 complex functions to preserve genomic integrity.