Chromosome segregation must occur without error each time a cell divides or else a genetic catastrophe occurs. When these catastrophic errors occur, an entire chromosome-and all of the genes it houses-will be present in daughter cells at one too many or one too few copy number (i.e. aneuploidy). Genome integrity at cell division relies on the autonomous action of a locus, termed the centromere, present on each chromosome. One centromere component is the master mitotic regulator, the Aurora B kinase. At the centromere, Aurora B monitors chromosomal connections to the microtubule-based mitotic spindle. Aurora B is the central player in a quality control mechanism called mitotic error correction. Aurora B has to be at the correct location on the chromosome, at the proper levels, and fully enzymatically activated by a regulatory modification to its closest binding partner, INCENP. Key information is lacking in all three of these areas. The specific sub-centromeric positioning of Aurora B is key to its mitotic error correction function, but there is currently onl a very low-resolution understanding of its positioning. Likewise, almost nothing is known about how the recruitment of Aurora B to centromeres is dynamically regulated in a chromosome autonomous fashion in response to chromosome/spindle attachment status. Further, it is known that the catalytic activity of Aurora B is activated only when bound to a phosphorylated form of its partner, INCENP, but precisely how this activation occurs remains unclear. We now propose perform experiments with the following specific aims: 1) To test the hypothesis that proper targeting of Aurora B to mitotic centromeres is dictated by a combination of chromatin features 2) To test the hypothesis that the PLK1 kinase modulates the levels of Aurora B in response to bipolar attachment to the mitotic spindle and 3) To test the hypothesis that phosphorylation of the Thr-Ser-Ser (TSS) motif of INCENP leads to structural and dynamic changes to Aurora B that fully activate the kinase. Using cell lines housing naturally-occurring neocentromeres that form on complex DNA, we will perform high-resolution genomic analysis of Aurora B complexes at mitotic centromeres. In addition, we will employ molecular genetic and cell biological approaches to directly interrogate the mechanisms underlying Aurora B-mediated error correction. Using solution-based biophysical measurements we will determine the changes that occur within the Aurora B molecule during its enzymatic activation, and determine the consequences of mutations in Aurora B that render it either constitutively active or inactive. Each of the three aims is designed to provide more mechanistic detail than the previous. In this way, the project is poised to produce significant insight into the molecular mechanisms underlying the key quality control pathway guarding the integrity of the genome at cell division.
Chromosomes must be transmitted equally during cell division or else there will be irreversible changes in chromosome/gene copy number. To avoid errors in this process, the Aurora B kinase is the prime quality control molecule. Our proposed studies are likely to substantially change our understanding of mitotic quality control with implications for deciphering fundamental genetic mechanisms in healthy cells and for defining the molecular roots of the defects in the Aurora B pathway that contribute to chromosome instability in tumor- derived cells.
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|Dawicki-McKenna, Jennine M; Langelier, Marie-France; DeNizio, Jamie E et al. (2015) PARP-1 Activation Requires Local Unfolding of an Autoinhibitory Domain. Mol Cell 60:755-768|