8 - Centromeres and Kinetochores Flashcards
What are kinetochores?
Kinetochores are the protein structures that anchor the microtubules of the spindle to the mitotic chromosomes at their centromeres
What is the role of kinetochores?
Enabling the spindle to bind to the chromosomes and so direct them to alignment on the metaphase plate axis and subsequently pull them apart. This requires highly directed pushing and pulling motions.
How do the kinetochores maintain genetic stability during mitosis?
• Binding to the centromere with great accuracy
o If the kinetochore linked the spindle not where the chromosomal cross is, the DNA would break when being separated
• Maintaining strong contact between the microtubules and the centromere during rapid polymerisation and depolymerisation of the microtubules
o Enables the spindles to move the chromosome around
• Proper kineotchore formation is the basis of the Spindle Assembly Checkpoint (SAC) which prevents the onset of anaphase until the chromosomes are properly aligned.
How conserved are kinetichores?
The structure and its function are largely conserved within eukaryotes (i.e. those that use it). However there are some differences between lower eukaryotes and higher eukaryotes (i.e. metazoans – animals).
You can see a large amount of homology between the two systems however, with the notable addition in budding yeast kinetochores of the Cbl3 complex, and there are also additional functional complexes wihtin the Ipl1 complex
What are the three layers of kinetochore structure?
• DNA Layer o The centromere and the centrosome – specialised nucleosomes • DNA Binding Layer o Cbl3 and Cbf1p in yeast o CCAN, CENP-B and CENP-C in metazoa • Microtubule Binding Layer o The Dam1/Ndc80 complexes in yeast o Ndc80/Ska complexes in metazoa
How many proteins are present in lower and higher eukaryotic kinetochores?
There are around 65 in cerevisiae and upwards of 80 so far identified in humans, with additions being made to each complex.
How many microtubules are found at the centromeres?
Higher eukaryotes also have a greater number of microtubule attachment sites, 15-20 microtubules can be recruited to each centromere as opposed to the single one found in cerevisiae, and the same applies to the number of nucleosomes involved.
What is a centromere?
The centromere is the point of primary constriction on the sister chromatids and marks the place where they are most closely linked. The chromosomes can be metacentric, acrocentric or submetacentric depending on whether the centromere is found in the middle of the chromosome, at the tip or somewhere in between respectively.
What is the centromere structure of budding yeast?
Budding yeast possess a point centromere, a single specialised nucleosome with a highly conserved centromere sequence. This consists of three sequences called CDE I, II and III that bind specifically to the specialised octamer.
What is the centromere structure of fission yeast?
Fission yeast have a more complex regional centromere than budding yeast, possessing a core sequence with satellite sequences on either side.
What is the centromere structure of hoomins?
In humans the core of the centromere is a repeating ‘α-satellite’ sequence that can stretch up to 5Mbp, bookended by regions of heterochromatin .
What epi-marks are found at the human centromere?
The canonical nucleosomes are marked with a unique combination of histone modification epi-marks; H3K9me2 and H3K9me3 (which are typically activating marks) and severe hypoacetylations (typically inhibitory). In humans it is these epimarks that defines the centromere rather than the sequence.
What is the structure of the satellite repeats in human centromeres?
Each ‘monomer’ section is an approximately 171bp sequence of which there are a varying number of variations of varying similarity to one another. Some but not all of these will have a CENP-B box that binds CENP-B for kinetochore formation.
These group into higher order repeats which can be made of 2-30 monomers, but are in this case made of four. These are found in arrays, in which 100s of higher order repeats are grouped. Each chromoseom in humans will have a different structure.
How do the structures on non-human metazoan centromeres compare?
Other regional centromeres often have greater diversity in sequence and structure, despite which the CCAN (constitutive centromere associated network) that binds to them is largely conserved.
What are centromeric nucleosomes?
These are also calles CENPA nucleosomes, because in these H3 is replaced by Centromere Protein A (CENPA).
How many CENPA nucleosomes are found within a centromere?
There is only ever one of these associated with a point centromere, but there are many associated with a regional centromere, generally interspersed with normal (canonical) histone octamers.
How do canonical nucleosomes bind DNA?
In normal nucleosomes, the DNA wraps around the octamer with 1 ¾ full turns, via a left helical superturn, 146 base pairs. The whole complex has a diameter of 10nm.
What is the structure of a centromeric nucleosome?
Centromeric nucleosomes are usually homotypic octamers, where both of the H3 subunits have been replaces with CENPA, however heterotypic ones where only one is replaced and tetramers where only one of each (including CENPA but not H3) is present.
The purpose of heterotypic/tetrameric nucleosomes is unclear, they may actually not be representative of in vivo action.
How do centromeric nucleosomes bind DNA?
Centromeric nucleosomes bind less DNA than normal ones, managing to bind only 121bp. This is due to the slight difference between CENPA and H3 – CENPA has one less turn in the N-terminal alpha helix and a shorter tail after it, which in H3 stabilises an extra length of DNA.
This leads to less efficient packing by the centromeric nucleosomes.
Describe the nucleosome occupancy profile of centromeric nucleosomes.
The centromeric nucleosome does, on every chromosome, give a distinctive profile in which the probability of finding one that would overlap with it drops to very little and there is a sharp change leading up to and down from the centromere region, which has consistent likelihood of being bound to the octamer.
This demonstrates the high affinity and specificity of the CENPA nucleosome to the centromere.
How are point centromeres organised in terms of their macrostructure?
Having only one centromeric nucleosome on each strand they form a cruciform arrangement where the centromere sequence forms two stem loops perpendicular to the chromosome. This leaves the CENPA nucleosome at the tip, where it connects to the microtubule via the kinetochore
What is the looping model of regional centromere arrangement?
The looping model suggests that it forms a series of cruciform structures, similar to a row of point centromeres.
What is the solenoid model of regional centromere arrangement?
The solenoid model suggests that each strand of DNA forms a single stranded solenoid on either side of the chromosome, with the centromeric nucleosome at the tip of each coil. The solenoid model is thought to be more in-keeping with the compaction habits of DNA.
How do the microtubules attach to the kinetochores?
The microtubules from the spindle attach to the kinetochores in prometaphase, often cycling through a variety of attachment conformations before the chromosome is correctly positioned.
Any of the incorrect attachments is sensed by Mad2, which will then arrest the cell cycle until the correct one is found.
What different microtubule attachments to the centromeres do not satisfy the spindle checkpoint?
- Non-telic – unattached
- Monotelic – only one spindle has attached
- Amphitelic – both spindles attached, but have not yet produced enough tension
- Syntelic – both kinetochores bound by microtubules from the same spindle
- Merotelic – one of the kinetochores is bound by microtubules from both spindle
What is lateral attachment?
The microtubules can also attach incorrectly by not approaching the kinetochore in a manner perpendicular to the kinetochore, an attachment for which kinesins are responsible.
With what should the microtubules bind to the kinetochore?
This is in contrast to end-on attachment, in which the microtubules do approach the kinetochore from a right angle, as shown in this electron tomography image.
What is the normal mechanism of how microtubules grow and shrink?
Microtubules can be elongated as they polymerise and shrink as they depolymerise. Their extension is simply dependent on a high concentration of αβ-tubulin dimers, and likewise a low concentration will lead to gradual depolymerisation.
