(APPLICANT?S ABSTRACT): The organization of eukaryotic replicons is both complex and poorly understood. Whereas regulatory mechanisms assure that chromosomes are duplicated only once per cell division, deviations from strict cell cycle control occur, often leading to the amplification of oncogenes in humans. Organisms that undergo developmentally-programmed gene amplification provide unique opportunities for understanding how replication origins can selectively escape cell cycle control. The Tetrahymena thermophila rDNA minichromosome is an excellent model for dissecting the role of cis-acting determinants, trans-acting factors and chromatin structure in DNA replication control. Tetrahymena rDNA is amplified 5000-fold within a single S phase during development, but is subsequently replicated once per cell cycle. Research in the prior granting period uncovered novel cisacting regulatory determinants for DNA replication, including one that may be dedicated solely to gene amplification.
Specific Aim 1 will further dissect the rDNA replicon, and determine the underlying role for newly discovered cis-acting elements in gene amplification and cell cycle-controlled DNA replication. Our biochemical studies have led to the discovery a complex regulatory pathway, in which cell cycle-regulated proteins (TIF 1-4) compete in vivo for binding to an essential rDNA replication determinant.
In Aims 2 and 3, we propose reverse genetic approaches to elucidate the role of these proteins and Tetrahymena ORC in rDNA replication control.
In Aim 4, we will investigate a developmentally-programmed switch that occurs during gene amplification to generate a strong replication fork barrier. These studies will include examining the role of cis-acting determinants in fork barrier formation, identifying proteins that mediate fork arrest, and exploring the role of chromatin remodeling in amplification of the rDNA minichromosome. The complexity of replicons in higher eukaryotes demonstrates the need to develop non-yeast model systems. Facile forward and reverse genetic approaches in Tetrahymena will be exploited to identify and explore the role of cis-acting determinants and trans-acting factors in the regulation of eukaryotic DNA replication, including underlying mechanisms for gene amplification.
| Sandoval, Pamela Y; Lee, Po-Hsuen; Meng, Xiangzhou et al. (2015) Checkpoint Activation of an Unconventional DNA Replication Program in Tetrahymena. PLoS Genet 11:e1005405 |
| Gao, Shan; Xiong, Jie; Zhang, Chunchao et al. (2013) Impaired replication elongation in Tetrahymena mutants deficient in histone H3 Lys 27 monomethylation. Genes Dev 27:1662-79 |
| Donti, Taraka R; Datta, Shibani; Sandoval, Pamela Y et al. (2009) Differential targeting of Tetrahymena ORC to ribosomal DNA and non-rDNA replication origins. EMBO J 28:223-33 |
| Mohammad, Mohammad; York, Randall D; Hommel, Jonathan et al. (2003) Characterization of a novel origin recognition complex-like complex: implications for DNA recognition, cell cycle control, and locus-specific gene amplification. Mol Cell Biol 23:5005-17 |
