What is your assay for sister-chromatid cohesion? - PubMed (original) (raw)

Review

What is your assay for sister-chromatid cohesion?

Frank Uhlmann. EMBO J. 2007.

No abstract available

PubMed Disclaimer

Figures

Figure 1

Figure 1

An assay for cohesin removal from chromosomes that revealed evidence for Scc1 cleavage by separase. Yeast whole-cell extract from metaphase-arrested cells (WE, lane 1) was separated into a supernatant (SU, lane 2) and chromatin fraction (CP, lane 3), which were kept on ice for the duration of the experiment. Scc1 was C-terminally tagged with an HA epitope to facilitate its detection by western blotting. An aliquot of supernatant and chromatin each were incubated for 10 min at 25°C to monitor unspecific Scc1 degradation under these conditions (lanes 4 and 5). Extracts were also prepared from an esp1-1 mutant yeast strain in which GAL1 promoter-driven overexpression of wild-type Esp1 had not (lane 6) or had been induced (lane 9). Note that Scc1 in these extracts did not carry an epitope tag. Aliquots of the Scc1-HA chromatin fraction were incubated in these extracts for 10 min at 25°C. After incubation in the esp1-1 extract, a small amount of full-length Scc1 leaked into the supernatant (lane 7), while most Scc1 remained associated with chromatin (lane 8). In contrast, after incubation in the Esp1-overexpressing extract, a significant portion of Scc1 was released from chromatin (lane 11) and was seen cleaved in the supernatant (lane 10).

Figure 2

Figure 2

Dual role of separase in triggering anaphase onset and mitotic progression. (I) After degradation of its inhibitor securin, separase is free to cleave cohesin, thereby triggering anaphase onset. Cohesin cleavage allows most sister sequences to separate, but not the budding yeast rDNA locus that remains interlinked by DNA catenation. (II) At the same time, separase causes downregulation of the PP2ACdc55 phosphatase. The mechanism for this is not yet understood, but involves an activity of separase independent of its proteolytic activity. This allows Cdk-dependent phosphorylation of Net1, the nucleolar inhibitor of the Cdc14 phosphatase, releasing active Cdc14. Initially, Cdc14 dephosphorylates targets important for anaphase spindle stabilisation and rDNA decatenation. (III) As Cdk activity declines, Cdc14 activates a positive feedback loop, the MEN, that sustains Cdc14 release. Cdc14 activity at low Cdk levels leads to completion of mitotic exit and cytokinesis.

Figure 3

Figure 3

Cohesin rings in sister-chromatid cohesion. (A) Two

s

tructural

m

aintenance of

c

hromosomes subunits Smc1 and Smc3, with their extended coiled coil, form much of the circumference of the ring, kept closed by an interaction between the Smc subunits both at a dimerisation interface known as ‘hinge' and by interaction of their ATPase head domains. Two additional subunits Scc1 and Scc3 associate with the Smc heads. Cohesin is loaded onto chromosomes by a loading factor, the Scc2/4 complex. This loading requires hydrolysis of ATP bound to the Smc head domains within the cohesin complex. Once loaded, cohesin translocates away from Scc2/4, in a manner consistent with it sliding along chromatin, and accumulates in regions of convergent transcriptional termination. (B) Establishment of sister-chromatid cohesion during DNA replication. Replication fork components are important for the establishment of sister-chromatid cohesion, in particular the proteins Eco1, Ctf4 and the RFCCtf18 complex. They might (a) help the replisome slide through cohesin rings, or (b) be involved in a reaction to re-assemble or stabilise cohesin around pairs of replication products in the wake of the fork.

Similar articles

Cited by

References

    1. Aboussekhra A, Biggerstaff M, Shivji MKK, Vilpo JA, Moncollin V, Podust VN, Proti M, Hübscher U, Egly J-M, Wood RD (1995) Mammalian DNA nucleotide excision repair reconstituted with purified protein components. Cell 80: 859–868 - PubMed
    1. Anderson DE, Losada A, Erickson HP, Hirano T (2002) Condensin and cohesin display different arm conformations with characteristic hinge angles. J Cell Biol 156: 419–424 - PMC - PubMed
    1. Arumugam P, Gruber S, Tanaka K, Haering CH, Mechtler K, Nasmyth K (2003) ATP hydrolysis is required for cohesin's association with chromosomes. Curr Biol 13: 1941–1953 - PubMed
    1. Azzam R, Chen SL, Shou W, Mah AS, Alexandru G, Nasmyth K, Annan RS, Carr SA, Deshaies RJ (2004) Phosphorylation by cyclin B–Cdk underlies release of mitotic exit activator Cdc14 from the nucleolus. Science 305: 516–519 - PubMed
    1. Bhat MA, Philp AV, Glover DM, Bellen HJ (1996) Chromatid segregation at anaphase requires the barren product, a novel chromosome-associated protein that interacts with topoisomerase II. Cell 87: 1103–1114 - PubMed

Publication types

MeSH terms

Substances

LinkOut - more resources