Centrioles have two key functions in animal cells: (1) They recruit pericentriolar materials to form centrosomes or microtubule-organizing center and (2) they serve as basal bodies that template the formation of cilia. These functions are critical for proper chromosome segregation, cell division, cell signaling, and cell cycle control. Deregulation of centrosome number contributes to genome instability, a hallmark of cancer. One evolutionarily conserved feature of the duplication process is centriole configuration, which cycles between the engaged state, where the two centrioles grow orthogonally to one another during S phase, and the disengaged state during late mitosis or early G1, where centrioles are no longer tightly opposed. Using Xenopus egg extracts and human cells, we have discovered that centriole disengagement occurs at late mitosis, and is mediated by polo-like kinase (Plk1) and separase. Only the disengaged centrioles, and not engaged ones, can be duplicated in the upcoming S phase. Plk1 and separase, therefore, license centrioles for duplication. I propose the following two aims to further understand the molecular involvement of these two enzymes in the disengagement process, and to identify the relevant substrates. In Aim 1, I will employ a two-step disengagement assay using Xenopus egg extracts to examine whether Plk1 metaphase activity is required for disengagement. Additionally I will test whether Plk1 and separase function independently, or in the same pathway to drive centriole disengagement. Furthermore, I will use an in vitro reconstitution centriole disengagement assay to determine whether Plk1 and separase are sufficient to trigger centriole disengagement, or whether additional activities are required. In Aim 2, I plan to identify Plk1 substrates through both a candidate approach, and an unbiased in vitro biochemical screen that we have previously validated. Separase and Plk1 are known to work together to remove the cohesin complex from chromosomes to facilitate chromosome segregation during anaphase, which coincides with centriole disengagement. Thus, I will determine if the cohesin complex can be the substrate of the disengagement activity by first examining the localization of hScc1 and other members of the cohesin complex by microscopy. I will also assess whether hScc1 and SAS2 (another member of the complex) are involved in centriole engagement, by RNAi-induced knockdown of these proteins followed by analysis of centriolar phenotypes with microscopy techniques developed in our laboratory. In addition, I will test whether a group of proteins recently identified through a proteomic analysis of human centrosomes, can function as separase and Plk1 substrates. For this purpose, I have generated several reagents for a biochemical screen to identify novel substrates of separase and Plk1, using In Vitro Expression Cloning (IVEC), a method that has been widely used to identify substrates of many biological enzymes.