Abstract
Accurate segregation of chromosomes during cell division is essential. The Dam1 complex binds kinetochores to microtubules and its oligomerization is required to form strong attachments. It is a key target of Aurora B kinase, which destabilizes erroneous attachments allowing subsequent correction. Understanding the roles and regulation of the Dam1 complex requires structural information. Here we apply cross-linking/mass spectrometry and structural modelling to determine the molecular architecture of the Dam1 complex. We find microtubule attachment is accompanied by substantial conformational changes, with direct binding mediated by the carboxy termini of Dam1p and Duo1p. Aurora B phosphorylation of Dam1p C terminus weakens direct interaction with the microtubule. Furthermore, the Dam1p amino terminus forms an interaction interface between Dam1 complexes, which is also disrupted by phosphorylation. Our results demonstrate that Aurora B inhibits both direct interaction with the microtubule and oligomerization of the Dam1 complex to drive error correction during mitosis.
Highlights
IntroductionThe Dam[1] complex binds kinetochores to microtubules and its oligomerization is required to form strong attachments
Accurate segregation of chromosomes during cell division is essential
A comparison of lysine Ca–Ca distances observed cross-linked together versus all possible lysine Ca–Ca distances from the structures showed a clear enrichment of distances o20 Å at 1% false discovery rate (FDR), indicating our method accurately reports on the native structure (Supplementary Fig. 2)
Summary
The Dam[1] complex binds kinetochores to microtubules and its oligomerization is required to form strong attachments. It is a key target of Aurora B kinase, which destabilizes erroneous attachments allowing subsequent correction. The kinetochore is a network of protein complexes that assemble on centromeric DNA and mediate the attachment of chromosomes to dynamic spindle microtubules (MTs). We previously found that phosphorylation of Dam1p S20 decreases the apparent affinity of the Dam[1] complex to MTs, whereas phosphorylation of the C-terminal Ipl[1] sites together has little effect[2] Both N- and C-terminal regions of Dam1p have been implicated in MT binding, their specific roles remain elusive. The specific regions involved in the MT-binding interface have not been identified and the localization of half of the subunits remains unknown
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