Identification of TICRR, a novel checkpoint and replication regulator

Abstract

The eukaryotic cell cycle refers to a sequence of events by which a cell duplicates its genomic DNA and divides into two daughter cells. Deregulation of the cell cycle can cause aberrant cell proliferation, as well as genomic and chromosomal instability, events that contribute to the development of cancer. Along with the machinery that promotes cell cycle progression, cells have evolved surveillance mechanisms, or checkpoints, that protect the cells from DNA lesions. Understanding the molecular mechanisms by which checkpoints act is of clinical relevance, since mutations in checkpoint components are often associated with human developmental disorders and cause a predisposition to cancer. A collection of 336 zebrafish (Danio rerio) lines, each carrying a recessive embryonic lethal mutation caused by a retroviral insertion, was screened for novel genes required for the ionizing radiation-induced G2/M checkpoint. Among the mutant lines that displayed a defect in checkpoint activation, two carry insertions in a novel gene that we have named ticrr (for TopBP1 interacting, checkpoint and replication regulator). The loss of ticrr impairs DNA replication and disrupts the S/M checkpoint, leading to premature mitotic entry of cells with partially replicated genomes and mitotic catastrophe. Therefore, Ticrr is a novel cell cycle regulator essential for genomic integrity with roles in replication as well as in the S/M and G2/M checkpoints. We have identified the human ortholog of Ticrr and showed that both the human and zebrafish Ticrr proteins associate with TopBP1, a protein with known roles in checkpoints and replication. We hypothesized that TICRR is required for pre-initiation complex assembly during replication initiation, in an analogous manner to the TopBP1 yeast ortholog Dpbl 1 and the yeast protein Sld3. Consistent with this model, we show that ticrr-deficiency disrupts chromatin association of pre-initiation complex, but not prereplication complex, components in the zebrafish. The work described in this thesis demonstrates the utility of zebrafish for performing genetic screens for cell cycle regulators. We have used human cell lines to complement our studies in the zebrafish and showed that taking advantages of the strengths that each system offers represents a powerful strategy to elucidate gene function.