The coordinated temporal regulation of eukaryotic replication origins assures that each segment of the genome is replicated once and only once per cell cycle. Chromosomes are under constant assault from intrinsic and extrinsic sources of genotoxic stress, generating lesions that promote the collapse of elongating replication forks or the formation of double-strand breaks. Eukaryotes have evolved elaborate checkpoint pathways that coordinate cell cycle progression with DNA repair. The master regulator of the intra-S phase DNA damage checkpoint, ATR, initiates a signaling cascade that stabilizes stalled replication forks and represses new origins from firing. Recent studies in the model eukaryote, Tetrahymena thermophila, have identified a sequence-specific DNA binding protein that represses origin activation early in S phase, and is further required for intra-S phase checkpoint activation. This factor, TIF1, lacks homology to known proteins in the checkpoint response, and is the only eukaryotic origin binding protein that has been shown to regulate replication timing. This project will exploit powerful reverse genetic approaches to study origin repression and checkpoint activation in Tetrahymena, with the goal of understanding how these two processes are integrated to promote genome stability during normal cell cycles and in response to genotoxic stress. Thus far, TIF1 has been shown to regulate initiation from the well-characterized ribosomal DNA (rDNA) replication origin. The mechanism of TIF1-mediated repression will be determined by assessing the effect of depleting TIF1 on the recruitment of conserved replication proteins to the rDNA replication initiation site. The role of TIF1 at non-rDNA origins will also be examined by studying replication timing at ~50 non-rDNA origins in wild type and TIF1-deficient strains, using a powerful single molecule approach, SMARD. The contributions of TIF1 and ATR to the repression of late firing origins during genotoxic stress will also be investigated. These studies will apply cutting edge technologies to address two important questions in chromosome biology: the poorly understood control of replication timing, and the coordinate regulation of DNA replication and repair. The early divergence of Tetrahymena from yeast and humans should serve as a reference point for understanding the evolution of the DNA damage checkpoint response. These studies will provide fundamental training in molecular biology and genomic-based approaches to graduate and undergraduate students, including the mentoring of under-represented minorities in science, including women. All reagents will be made readily available to colleagues, including members of the ciliate molecular biology community. The findings of these studies will be disseminated by the PI and students at local and national meetings, as well as be published in open access journals.
