Person:

Hinshaw, Stephen

The cohesin ring holds newly replicated sister chromatids together until their separation at anaphase. Initiation of sister chromatid cohesion depends on a separate complex, Scc2NIPBL/Scc4Mau2 (Scc2/4), which loads cohesin onto DNA and determines its localization across the genome. Proper cohesin loading is essential for cell division, and partial defects cause chromosome missegregation and aberrant transcriptional regulation, leading to severe developmental defects in multicellular organisms. We present here a crystal structure showing the interaction between Scc2 and Scc4. Scc4 is a TPR array that envelops an extended Scc2 peptide. Using budding yeast, we demonstrate that a conserved patch on the surface of Scc4 is required to recruit Scc2/4 to centromeres and to build pericentromeric cohesion. These findings reveal the role of Scc4 in determining the localization of cohesin loading and establish a molecular basis for Scc2/4 recruitment to centromeres. DOI: http://dx.doi.org/10.7554/eLife.06057.001

Summary The ring-shaped cohesin complex brings together distant DNA domains to maintain, express, and segregate the genome. Establishing specific chromosomal linkages depends on cohesin recruitment to defined loci. One such locus is the budding yeast centromere, which is a paradigm for targeted cohesin loading. The kinetochore, a multiprotein complex that connects centromeres to microtubules, drives the recruitment of high levels of cohesin to link sister chromatids together. We have exploited this system to determine the mechanism of specific cohesin recruitment. We show that phosphorylation of the Ctf19 kinetochore protein by a conserved kinase, DDK, provides a binding site for the Scc2/4 cohesin loading complex, thereby directing cohesin loading to centromeres. A similar mechanism targets cohesin to chromosomes in vertebrates. These findings represent a complete molecular description of targeted cohesin loading, a phenomenon with wide-ranging importance in chromosome segregation and, in multicellular organisms, transcription regulation.

Chromosome segregation during mitosis requires that two forces act on DNA: one that pulls chromatids away from each other and towards presumptive daughter cells, and another that holds chromatids together and resists the first force. These forces converge on the centromere, a specialized region of the chromosome upon which is built the kinetochore, a multi-protein apparatus that connects spindle microtubules to DNA. Kinetochores participate in a signaling pathway that recruits the molecular agents of sister chromatid cohesion, protein complexes called cohesins, to centromeres. The accumulation of cohesin complexes at centromeres is required to resist spindle forces until anaphase, thereby ensuring efficient chromosome segregation.

In this thesis, we describe the molecular events that conspire to recruit cohesin complexes to centromeres. This work presented here shows that the protein complex that loads cohesin onto chromosomes recognizes centromeres, a function that depends on Dbf4-dependent kinase (DDK) phosphorylation of the Ctf19 kinetochore protein. We also describe the reconstitution and analysis of factors that ensure proper cohesin loading. These studies provide a molecular explanation for the connection between sister centromeres and its regulation during the cell division cycle.