Kinetochore-microtubule error correction for biorientation:lessons from yeast

Accurate chromosome segregation in mitosis relies on sister kinetochores forming stable attachments to microtubules (MTs) extending from opposite spindle poles and establishing biorientation. To achieve this, erroneous kinetochore-MT interactions must be resolved through a process called error correction, which dissolves improper kinetochore-MT attachment and allows new interactions until biorientation is achieved. The Aurora B kinase plays key roles in driving error correction by phosphorylating Dam1 and Ndc80 complexes, while Mps1 kinase, Stu2 MT polymerase and phosphatases also regulate this process. Once biorientation is formed, tension is applied to kinetochore-MT interaction, stabilizing it. In this review article, we discuss the mechanisms of kinetochore-MT interaction, error correction and biorientation. We focus mainly on recent insights from budding yeast, where the attachment of a single MT to a single kinetochore during biorientation simplifies the analysis of error correction mechanisms.</p

The nucleoporin ALADIN regulates Aurora A localization to ensure robust mitotic spindle formation

The formation of the mitotic spindle is a complex process that requires massive cellular reorganization. Regulation by mitotic kinases controls this entire process. One of these mitotic controllers is Aurora A kinase, which is itself highly regulated. In this study, we show that the nuclear pore protein ALADIN is a novel spatial regulator of Aurora A. Without ALADIN, Aurora A spreads from centrosomes onto spindle microtubules, which affects the distribution of a subset of microtubule regulators and slows spindle assembly and chromosome alignment. ALADIN interacts with inactive Aurora A and is recruited to the spindle pole after Aurora A inhibition. Of interest, mutations in ALADIN cause triple A syndrome. We find that some of the mitotic phenotypes that we observe after ALADIN depletion also occur in cells from triple A syndrome patients, which raises the possibility that mitotic errors may underlie part of the etiology of this syndrome

The CRISPR associated protein Cas4 Is a 5' to 3' DNA exonuclease with an iron-sulfur cluster

The Cas4 protein is one of the core CRISPR-associated (Cas) proteins implicated in the prokaryotic CRISPR system for antiviral defence. Cas4 is thought to play a role in the capture of new viral DNA sequences for incorporation into the host genome. No biochemical activity has been reported for Cas4, but it is predicted to include a RecB nuclease domain. We show here that Cas4 family proteins from the archaeon Sulfolobus solfataricus utilise four conserved cysteine residues to bind an iron-sulfur cluster in an arrangement reminiscent of the AddB nuclease of Bacillus subtilis. The Cas4 family protein Sso0001 is a 5' to 3' single stranded DNA exonuclease in vitro that is stalled by extrahelical DNA adducts. A role for Cas4 in DNA duplex strand resectioning to generate recombinogenic 3' single stranded DNA overhangs is proposed. Comparison of the AddB structure with that of a related bacterial nuclease from Eubacterium rectales reveals that the iron-sulfur cluster can be replaced by a zinc ion without disrupting the protein structure, with implications for the evolution of iron-sulfur binding proteins.Publisher PDFPeer reviewe

Structural similarities between AddB, Cas4 and a putative exonuclease DUF3799.

<p>All three structures show the same arrangement of four cysteine residues forming a metal ligand, coupled with a RecB nuclease domain. However, unlike AddB (PDB: 3U44) and Cas4, DUF3799 (PDB: 3L0A) has a zinc ion (green sphere) coordinated with the cysteines instead of iron. The cartoons on the left show the conserved residues implicated in nuclease activity and FeS cluster binding.</p

Cas4 family proteins are metal dependent nucleases.

<p>A. Sso0001 (0.6 µM) cleaves the ssDNA oligonucleotide 15T (1 µM) in the presence of either Mg²⁺ or Mn²⁺ but the ssRNA oligonucleotide 20U (1 µM) only in the presence of Mn²⁺. Both oligonucleotides were 5′-end labelled with a 5′-fluorescein moiety. Reactions were carried out at 75°C for 10 min. B. The Sso0001 D99A variant does not cleave ssDNA oligonucleotide 15T (1 µM) in the presence of Mg²⁺ ions. Assays were carried out as in panel A with 0.6 or 1.2 µM of the relevant protein. Reactions were carried out at 75°C for 10 min. C. Circular phiX174 virion ssDNA (60 nM) was incubated with 1.2 µM wild-type or 3 µM D99A Sso0001 for 10, 20 and 40 min at 55°C in the presence of MgCl₂. No degradation of the DNA was observed, suggesting that Sso0001 requires a ssDNA end for activity. The control lane (C) was incubated in the same conditions in the absence of enzyme. dsDNA size markers (m) are indicated.</p

Cas4 family proteins are specific for single-stranded DNA and stalled by an internal extrahelical fluorescein.

<p>The left side of the gel shows degradation of a 31nt oligonucleotide with an internal fluorescein-dT at position 20. The right hand side shows this oligonucleotide in a 31bp DNA duplex (both at 1 µM final concentration). The assay was carried out with 0.6 µM Sso0001 at 55°C for time points of 0, 1, 2, 3, 5, 10, 20, 30 and 40 min in 10 mM Mg²⁺. Control lanes (c) lacked added Sso0001 enzyme.</p