SUMMARY Our goal is to identify how Polo-like kinase 1 (Plk1) signals at the human centromere to maintain genome integrity. In mitosis, the kinetochore (KT) is assembled at the centromere and this complex is a major focus of Plk1 activity. The KT is a 100 nm-structure assembled on the centromere that attaches chromosomes to the mitotic spindle. Using super-resolution techniques, we have discovered that the bulk of Plk1 localizes to the centromere, directly on chromatin >50 nm from the outer KT. Our long-term goal is to delineate Plk1 partners, substrates, and timing of its activities to maintain faithful chromosome segregation. We are currently focused on activities at the centromere, building on our key findings: Bloom Syndrome RecQ Helicase (BLM) directly or indirectly mediates chromosome arm dislocation and centromere unwinding that occur with loss of Plk1 activity; Plk1 regulates nascent transcripts on chromatin in mitosis and phosphorylates the N-terminus of Centromere Protein C (CENP-C), both known to maintain KT integrity. Our central hypothesis is that a discrete centromere pool of Plk1 stabilizes the mitotic centromere and operates through inactivating helicases, by supporting transcription, and by maintaining CENP-C function. In Aim 1, we will map the spatial environments of Plk1 that contribute to its functions along the KT-centromere axis. We will test the idea that a chromatin-localized pool of Plk1 targets substrates and mediates activities separately from a KT-localized pool, to regulate faithful chromosome segregation. Aim 2 will test how Plk1 dampens BLM helicase function to maintain centromere integrity against mitotic pulling forces. To do this, we will identify Plk1 phosphorylation sites on the BLM protein, determine the role of Plk1 on BLM localization, and evaluate how phosphorylation modulates its helicase activities in biochemical assays and cells. Aim 3 will identify the role of Plk1 on CENP-C stabilization via direct phosphorylation and transcriptional regulation. We have already mapped Plk1 phosphorylation sites on CENP- C and have discovered that Plk1 regulates mitotic transcription. Towards this end, we will functionally analyze the CENP-C phosphorylation events and identify the functional effect of Plk1 on RNA polymerases and mitotic RNAs. Together, this work will reveal how Plk1 operates regionally in the KT to maintain genomic integrity.

NARRATIVE Genome instability is a common feature of multiple human diseases. This project focuses on the signaling mechanisms of Polo-like kinase 1 at chromosome centromeres that are required to maintain stability. By understanding chromatin-based signals, it will be possible to develop a basic mechanistic understanding of disease.