Project Summary A central question in the study of cell cycle regulation is, how the cell cycle transitions are controlled to allow the transition to the next cell cycle stage at a right time? Such control mechanisms are critical for maintaining genome integrity because premature transition from one stage to the next stage of the cell cycle can lead to aneuploidy and, hence, cell death. Four major cell cycle transition points, the G1/S transition, the G2/M transition, the metaphase/anaphase transition, and the mitosis/cytokinesis transition, are under stringent control, but how they are regulated at the molecular level in T. brucei, a protozoan parasite and the causative agent of human sleeping sickness, remains poorly understood. Cyclin-dependent kinases (CDKs) are key regulators of the cell cycle transitions in eukaryotes, and T. brucei employs the CDK-related kinase CRK1 to control the G1/S transition, CRK2 to promote S-phase progression, and CRK3 to govern the G2/M transition. The roles of CRK1 in controlling the nuclear events in promoting the G1/S transition and the role of CRK2 in regulating S-phase progression have been recently revealed by us. However, the molecular mechanisms underlying the G2/M transition and the metaphase/anaphase transition are still elusive. Further, it is generally accepted that T. brucei lacks several cell cycle checkpoints, including the spindle assembly checkpoint, and it is unclear how T. brucei recognizes the genomic errors occurred during the cell cycle and prevents erroneous genome duplication and premature genome segregation. Our recent discovery of a novel DNA damage- induced metaphase checkpoint suggests a kinetochore-based checkpoint machinery in T. brucei, but the underlying mechanism remains largely unknown. The current proposal aims to understand the control of the G2/M transition by CRK3-mediated regulation of the chromosomal passenger complex, the control of metaphase/anaphase transition by CRK3-mediated regulation of kinetochore proteins, and the mechanism of the metaphase checkpoint mediated by KKIP5 and its associated proteins. The outcomes from these investigations not only will have important biological significance, but also could provide novel targets for anti- trypanosomiasis chemotherapy.