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Sister-chromatid cohesion makes it easy to biorient

Sister-chromatid cohesion makes it easy to biorient. With sister cohesion. Without sister cohesion. Sister-chromatid cohesion by DNA catenation. Cutting Scc1 here triggers sister separation in anaphase. Cohesin is for cohesion. Smc1-Smc3 dimer. Smc1-Smc3 dimer + Scc1 and Scc3. cutting

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Sister-chromatid cohesion makes it easy to biorient

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  1. Sister-chromatid cohesion makes it easy to biorient With sister cohesion Without sister cohesion

  2. Sister-chromatid cohesion by DNA catenation

  3. Cutting Scc1 here triggers sister separation in anaphase Cohesin is for cohesion Smc1-Smc3 dimer Smc1-Smc3 dimer + Scc1 and Scc3

  4. cutting Scc1 here triggers sister separation How does cohesin hold sisters together?

  5. Testing for topological interactions between cohesin and a circular minichromosome sucrose gradient native agarose gel Ivanov and Nasmyth (2007) Mol. Cell27, 300

  6. Minichromosome dimers are held together by cohesin Ivanov and Nasmyth (2007) Mol. Cell27, 300

  7. cross- link protein ends cross- link protein ends monomer circles dimers! The latest test of the two major models: Haering et al., 2008 If it’s physical contact If it’s a topological link denature denature monomer circles monomer circles

  8. Putting on cohesin Loading Scc2/4 + Cohesin ATPase activity Establishment DNA replication Eco1 Wpl1 G1 S phase G2 and M

  9. Affinity of cohesin for chromatin iFRAP of GFP-tagged cohesin subunit SA1 Scc3 Scc1 Scc3 Gerlich et al (2006) Curr. Biol. 16, 1571

  10. Affinity of cohesin for chromatin Gerlich et al (2006) Curr. Biol. 16, 1571

  11. How does cohesin get loaded? Gruber et al. (2006) Cell127, 523.

  12. Does cohesin loading require opening the SMC-Scc1 connection? Normal function! Normal function! Normal function! Gruber et al. (2006) Cell127, 523.

  13. Replacing the SMC hinge blocks cohesin binding to chromosomes Gruber et al. (2006) Cell127, 523.

  14. The balance of cohesin loading, release and establishment Establishment Loading Scc2-4 + ATP Ac Eco1 DNA replication Wpl1 Wpl1 Wpl1 Wpl1 Chan et al. (2012) Cell150, 961. Release Release G1 S

  15. Go through the hoop, or flip it over?

  16. Sister chromatid condensation and resolution Sumner, A.T. Chromosoma 1991, 100:410

  17. We know next to nothing about chromosome condensation 400-700 nm 30 nm Belmont Curr. Opin. Cell Biol. 2006, 18:632

  18. Condensin is required for normal mitotic chromosome assembly in Xenopus extracts Control antibody Anti-condensin antibody Hirano and Mitchison Cell 1994, 79:449-458

  19. The Condensin complex

  20. Condensin at the chromosome axis in metaphase DAPI Smc2 DAPI Topo II Topo II Smc2 Fluorescence microscopy Electron microscopy: immunogold of Smc2 Kireeva et al. J. Cell Biol. 2004, 166:775

  21. How does condensin organize chromosomes? Glue Ring Floral arrangement Cuylen and Haering Trends Cell Biol. 2011, 21:552

  22. Evidence for a condensin ring! Bgl II TEV protease Cuylen et al. Nat. Struct. Mol. Biol. 2011, 18:894

  23. Artificial condensin ring cutting makes the sister chromatids floppier Cuylen et al. Nat. Struct. Mol. Biol. 2011, 18:894

  24. Condensin is not required for condensation in human cells (?!) CAP-D2 (condensin I) RNAi SMC2 (both condensins) RNAi CAP-D3 (condensin II) RNAi CAP-D2 & CAP-D3 RNAi Control RNAi isotonic treatment hypotonic treatment Nocodazole, hypotonic treatment Hirota et al. J. Cell Sci. 2004, 117:6435

  25. Condensin is required for normal anaphase Control RNAi CAP-D2 (condensin I) RNAi CAP-D3 (condensin II) RNAi SMC2 (both condensins) RNAi Hirota et al. J. Cell Sci. 2004, 117:6435

  26. Condensin might also help organize sisters for decatenation by topoisomerase II Topo II Cuylen and Haering Trends Cell Biol. 2011, 21:552

  27. The ‘Prophase pathway’: In animal cells, cohesin is removed from chromosome arms in early mitosis Polo kinase removed Aurora B kinase removed Both kinases removed control DNA cohesin Losada et al. Genes Dev. 2002, 16:3004

  28. Cohesin removal from arms is required for sister resolution control Both kinases removed Losada et al. Genes Dev. 2002 16:3004

  29. Phosphorylation of a cohesin subunit is required for removal of arm cohesin in prophase SA2 in humans [wild type] [Nonphosphorylated mutant] Hauf et al. PLoS Biol. 2005, 3:e69

  30. We still know next to nothing about mitotic chromosome structure Compaction of DNA from same sister Resolution of DNA strands from different sisters 3. Coordination of condensin and cohesin 4. How do Cdks regulate all this?

  31. remain attached when microtubule is growing • and shrinking growing shrinking Kinetochore The many functions of the kinetochore Microtubule - - attach sister chromatid to microtubule plus end • allow regulation of attachment to ensure • biorientation • send signals that block anaphase • when attachment is incorrect + • generate force to move chromosomes Centromere DNA

  32. Kinetochores have many parts outer inner budding yeast animals

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