1 Walther Flemming: ‘Zellsubstanz, Kern und Zelltheilung.’ Leipzig, 1882 Cohesin, Chromosomes and Cancer David Hansemann (1890): Über asymmetrische Zelltheilung in Epithel Krebsen und deren biologische Bedeutung Virchow’s Arch. Pathol. Anat. 119, 299-326 'Besonders wichtig erscheint die asymmetrische Theilung vom Standpunkte des Panmerismus, […], dass die biologischen Eigenschaften einer Zelle an bestimmte geformte Elemente der Zelle gebunden sind,…' 'Es ist also wohl möglich, dass stellenweise eine Verzögerung der Längsspaltung eingetreten war.' Aneuploidy in a human colon cancer human karyotype colon cancer
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Walther Flemming: ‘Zellsubstanz, Kern und Zelltheilung.’ Leipzig, 1882
Cohesin, Chromosomesand Cancer
David Hansemann (1890):Über asymmetrische Zelltheilung in Epithel Krebsen und deren biologische BedeutungVirchow’s Arch. Pathol. Anat. 119, 299-326
'Besonders wichtig erscheint die asymmetrische Theilung vom Standpunkte desPanmerismus, […], dass die biologischen Eigenschaften einer Zelle an bestimmtegeformte Elemente der Zelle gebunden sind,…'
'Es ist also wohl möglich, dass stellenweise eine Verzögerung der Längsspaltungeingetreten war.'
Aneuploidy in a human colon cancer
human karyotype
colon cancer
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Lengauer et al. (1997)
Centromere 7Centromere 18
HT29HCT116
Genetic instability in colorectal cancer
• 85% of colocrectal cancers display chromosomal instability(as do most other solid tumours)
• a high degree of aneuploidy is taken as poor prognosis- evolution in new environment- resistance against treatment
• chromosomal instabilitiy can be seen in the earliest detectablelesions (2 mm adenomas)
Chromosomal instability: Facts
Does chromosomal instability cause cancer?
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Effect of taxol in chemotherapy might depend on the checkpoint status
How important is tetraploidy as route to aneuploidy?
spontaneous non-disjunction in immortalized human keratinocytes (N/TERT-1)
leads to cytokinesis regression and binucleate formation
Shi and King, 2005
tetraploid p53- mouse mammary epithelial cells (transient cytochalasin B
treatment), but not diploid controls, gave rise to tumours in nude mice
Pellman, 2005
Tetraploidy as a route to aneuploidy (IV)
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The cohesin cycle
Scc2/4
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Milutinovich and Koshland 2003
Campbell and Cohen-Fix 2002
Haering and Nasmyth, 2003
How does cohesin hold together sister chromatids?
A topological interaction between cohesin and DNA
circular minichromosome
loss of the cohesin-DNA interaction by:
1 cleavage of Scc1 (separase)
2 engineered cleavage of Smc3 (TEV protease)
3 restriction of the minichromosome
Ivanov and Nasmyth (2005) Cell 122, 849-860
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The Smc 'heads' are ABC ATPases that dimerize upon ATP binding:
ATP BindingWalker A: GxxGxGKS/TWalker B: hhDExDATP HydrolysisC-motif: LSGG
P. furiosus SMC:(Hopfner, 2004)
ATP Binding ATP HydrolysisDNA
ATP Binding ATP HydrolysisDNA
Scc2/4
10
50 nm
Atomic force microscopy reveals a head-hinge interaction
Cohesin Smc1/3 dimer:
opening ofheads or hinge?
Replisome
Replisome
+
Replisome
2. Cohesin re-assembly after fork passage
1. Replication fork sliding through the cohesin ring
a)
b)
And what happens during DNA replication?
Identification of replication products for segregation in mitosis
Biochemistry is easy with soluble proteins,but what does cohesin look like bound to chromosomes?
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DNase digest
M=S N0(6 Rs)/(1-v2 ).
Siegel and Monty, 1966
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why does a stable Scc1 cleavage productcause chromosome loss?
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Sister chromatid cohesion factors in congenital disorders
human Scc2 (NIPBL): Cornelia de Lange syndrome
human Eco1 (ESCO2): Roberts syndrome
Hierarchical folding models of a chromosome
‘scaffold’?
A proteinaceous chromosome scaffold?
• salt extraction ofnon-histone proteins• visualisation ofchromosome scaffoldby electron microscopy
U. Laemmli
scaffold after chemicaltreatment, is there a stablescaffold in live cells?
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Poirier and Marko, 2002
Newt chromosome between micro-pipettes
10 μm
+ D
Nase
Mitotic chromosomes are chromatin networkswithout a mechanically contiguous protein scaffold
Studying protein dynamics on chromosomes byFluorescense Recovery After Photobleaching (FRAP)
Topo
1. Topoisomerase II fused to Green Fluorescent Protein (GFP)2. Photobleaching of a defined region within the nucleus3. Does Topo II-GFP from the surrounding replenish fluorescense in the bleached area?
Answer, Yes: Topo II is not stably bound to chromosomes, but is dynamic and mobile