Eukaryotic genome organisation and cell cycle Flashcards
(88 cards)
1
Q
Tell me the following about eukaryotic DNA…
- Number of base pairs
- Length of base pair
- Length of haploid genomic DNA stretched out
A
- Human genome has 3 billion base pairs (3 x 109 bp)
- Each base pair is 0.34 nm long (0.34 x 10-9m/bp)
- Length of haploid genomic DNA stretched out =
- (3 x 109) x (0.34 x 10-9m) = 1 m
2
Q
How much DNA is found in a human chromosome?
A
19 to 73 mm (22 pairs plue XX or XY)
But it’s condensed to fit into cell nucleus (5 to 10 um in diameter)
3
Q
How can nucleosomes be seperated?
A
By incubation with DNase enzymes
4
Q
What are the histone proteins found in a nucleosome?
A
5
Q
The four core histones have what protruding from the nucleosome?
A
The four core histones have N-terminal tails protruding from the nucleosome
6
Q
Whats the role of the H1 histone?
A
It helps with packing the chromatin into further fibres
7
Q
How can histone N-terminal tails be modified?
A
By phosphorylation, acetylation methylation
8
Q
What are nucleosomes inportant for?
What happens if they are packed too tightly?
A
This is important for regulation of gene expression and condensing chromosomes during mitosis
if packed too tightly, no other factors can “interrogate” the chromosome and transcription is silenced
9
Q
What happens in the interphase step if the cell cycle?
A
Gene expression
Genome amplification
10
Q
What happens in the M phase of the cell cycle?
A
Mitosis
segregation of chromosomes (cytokinesis)
11
Q
Whats Chromatin?
What is it used for?
A
The complex of histones, non-histone proteins and nuclear DNA. Chromatin allows for the DNA compaction and is involved in the regulation of all DNA activites
12
Q
What kind of proteins are non-histone proteins?
A
Non-histone proteins are usually structural or regulatory proteins which effect chromatin structure and usually effect enzymatic activity within chromatin
13
Q
What are nucleosomes?
A
The basic structural unit of chromatin
14
Q
What makes up the histone octamer?
A
The Dimers H3-H4 and H2A-H2B are repeated twice which makes up the histone octamer
15
Q
What do each of the core histones (from the octamer) possess?
A
Two functional domains; amino-terminal tail and histone fold (not only histones which have this domain, found in some transcription factors.
16
Q
What do histone folds interact with?
A
Histone folds interact with each other, allowing formation of dimers (via the ‘handshake’ interaction)
17
Q
How many bp from the dsDNA winds around the histone core?
How many turns is this?
A
147 bp
It forms slightly less than two turns
18
Q
How does the linker histone, H1 work?
A
H1 bind both to DNA and the nucleosome core
They can change the path of DNA that exits the nucleosome. Hence, it affects the linker DNA accessibility, organisation of higher order chromatin fibre and chromatin compaction
19
Q
What do Hooks on the histone represent?
A
Places where DNA interacts directly with histones
20
Q
Whats the name of the model that explains the chromatin structure?
A
The ‘Zig-Zag’ model
21
Q
During interphase, how is chromatin arranged?
What helps to form these structures?
What two components were identified so far?
A
in loops
Architectural proteins are involved in the formation of these loops
The two components identified so far are; Protein complex called cohesin and a protein called CTCF
22
Q
What does cohesin and CTCF form?
A
Chromatin loops
23
Q
The size of the chromatin loops may change, what mechanism do chromatin loops grow via?
A
The loop extrusion mechanism
24
Q
during the loop extrusion mechanism, what are the chromatin fibres help together by?
What is the size of the loop regulated by?
A
Cohesin and the size of the loop is regulated by CTCF
25
IS loop extrusion a dynamic process?
yes, which requires ATP
26
What does loop extrusion help to do?
Position specific DNA sequences in areas of chromatin that are transcriptionally either active or inactive
27
When do loops stop growing?
When CTCF comes into close proximity with cohesin
All structurally regulated
28
How are the loops of chromatin further organised into higher order organisation of chromatin?
Further loops can form from pre-existing loops (TADs)
Further hierarchical organisation of interphase chromatin: groups of chromatin loops form **Topologically Associating domain (TADs)**
29
Whats are TADs?
TADs are grouped into compartments; compartments may be **transcriptionally active (type A) or inactive (type B)**
compartments belong to individual chromosomal territories, which are occupied by single chromosomes decondensed after mitosis
30
How can chromosomal territories be visualised?
using fluorescent staining
A technique to paint chromosomes using multi-colour FISH (spectral karyotyping) helps to visualise entire chromosomes
31
Tell me some features of chromosomes territories?
* some chromosomes localize toward the periphery, often touching the nuclear membrane, whereas others are located toward the center of the nucleus
* recurrent clusters of chromosomes. For example, in mouse lymphocytes, chromosome 12 often sits next to chromosome 14, which in turn is adjacent to chromosome 15, thereby forming a triplet cluster
* there are large areas of chromosomal identity between different species that have been maintained throughout evolution
* patterns of chromosome arrangement are specific to both cell type and tissue type
* chromosome territories can reposition in disease
32
Conclusions...
* In eukaryotic chromatin DNA is wrapped around histone octamers (nucleosomes)
* During interphase chromatin fibres are arranged in loops. Cohesins and CTCF proteins define boundaries of most of these loops.
* Looping of chromatin fibres has a function in chromatic compaction and in the regulation of gene expression.
* Chromatin loops are organised in Topologically associating Domains (TADs)
* TADs are organised into compartments, which can be transcriptionally active or inactive
* Compartments form Chromosome Territories within interphase nuclei
* Position of a gene within these structures affects the activity of that gene.
* Genome organization depends on an organism, cell type (tissue), stage of development, cell cycle phase, current physiological status (e.g. stimuli from environment, stress) and is disturbed in many pathological states.
33
Label this mitotic chromosomes
34
In a metaphase chromosome, how much DNA does an individual chromatid contain?
one
35
What are condensins and what are the similar to?
Condensin are multiprotein complexes; in their structure they are similar to cohesins
36
Tell me the types of condensins and their roles
There are 2 types of condensins: **Condensin 1** and **Condensin 2**
They have similar structures, but different complexes. They have different roles in formation of mitotic chromosomes
* Condensin 1 forms small loops formations
* condensin 2 binds small loops together to form bigger loops.
37
are interphase chromatin and mitotic chromosomes organised differently?
yes
38
What happens during mitotic chromosome assembly?
* interphase loops, TADs and compartments disappear
* DNA is folded into a linar array of continuous chromatin loops, perhaps attached to a scaffold
* This array is compressed to form a mitotic chromosome
39
What do cohesins play an important role in?
The organisation of interphase genome
Condensins play equally important role duirng mutosis
40
How are centomeres of mitotic chromosomes established?
epigenetically
41
What are the important events of the cell cycle?
42
What are the phases of the cell cycle ?
43
What occurs in the **G1 phase?**
* cell grow
* cells can stay here or 'sleep' (all depends on cell type and environment as to whether it goes onto cell cycle or not)
44
What occurs in the **S phase?**
DNA is replicated
45
What occurs in the **G2 phase?**
* cells prepare for mitosis
* Decide whether to commit to chromosome segregation
46
What occurs in the **M phase?**
* mitosis
* cytokinesis
47
Is the length of the cell cycle the same in all species?
Why may it vary?
no
* Cell cycle timing and structure varies in different cells and organisms
* Adapt cell cycle to needs (length and presence of individual stages of cell cycle)
48
What does the fidelity of cell reproduction depend on?
The regulatory mechanisms that ensure that the events of the cell cycle occur in the correct order
49
What do eukaryotic cells contain that control the timing and coordination of cell cycling events?
A complex regulatory network called the **cell-cycle control system**
50
Tell me some characteristics of the cell cycle control system?
* robust
* reliable biochemical timer
* Highly adaptable
* can be modified to suit specific cell types
51
What are cell cycle **checkpoints?**
Cell cycle checkpoints may arrest the cycle at three different transitions, if something goes wrong. They work like binary switches that launch events in a complete, irreversible fashion
52
Where on the cell cycle are the three transition checkpoints?
What occurs in each transition?
* **Start –** major transition at the entry into the cell cycle in mid to late G1; progression past this point is prevented if cell growth is insufficient, DNA is damaged or other preparations are not complete. Cells, instead of arresting, enter a prolonged nondividing state, if the conditions to pass this checkpoint are not met.
* **G2/M checkpoint –** regulatory transition where entry into M phase can be controlled by various factors, such as DNA damage or the completion of DNA replication
* **Metaphase-to-Anaphase transition –** transition during M phase where the initiation of the sister chromatid separation can be blocked, if chromosomes are not properly attached to microtubules of the mitotic spindle
53
what does cyclin regulate the activity of?
Cdk
54
When cyclin forms a complex with Cdk, what happens?
The protein kinase is activated to trigger specific cell-cycle events.
Without cyclin, Cdk is inactive
55
Oscillations in Cdk activity during the cell cycle are primarily due to what?
They are primarily due to changes in the amounts of cyclins. Different types of cyclins are produced at different cell-cycle phases. This results in the periodic formation of distinct cyclin-Cdk complexes that trigger different cell-cycle events
56
Where are the cyclin-Cdk complexes in the cell-cycle control system?
57
Tell me about how levels of cyclin and Cdk change during the cell cycle?
* The concentrations of cyclins oscillate during the cell cycle
* The concentrations of Cdks do not change and exceed cyclin amounts
58
What regulates cell cycle progression?
Cyclins regulate cell cycle progression, as cyclin levels change and Cdk levels don't
59
What do cyclins and Cdk provide to the cell cycle?
Cyclin delivers the regulatory mechanisms
Cdk provides the enzymatic activity needed for the regulation
60
What type of regulation is seen through the cell cycle and why?
Phosphodependent regulation is seen through the cell cycle, as kinase phosphorylate
The cell cycle is phosphoregulated by Cdks
61
What types of cyclins are there?
* G1 cyclins
* G1/S cyclins
* S cyclins
* M cyclins
62
Whats the role of **G1 cyclins?**
cyclins that bind and activate Cdks that stimulate entry into a new cell cycle at Start; their concentration depends on the rate of cell growth or on growth-promoting signals rather than on the phase of the cell cycle.
63
Whats the role of **G1/S cyclins?**
cyclins that activate Cdks that stimulate progression through Start, resulting in a commitment to cell-cycle enrty; their concentration peaks in late G1.
64
Whats the role of **S cyclins?**
cyclins that activate Cdks necessary for DNA synthesis; their concentrations rise and remain high during S phase, G2 and early mitosis; they contribute to the control of some early mitotic events.
65
Whats the role of **M cyclins ?**
cyclins that activate Cdks necessary for entry into mitosis; their concentration rises at the approach to mitosis and peaks in metaphase.
66
How do different cyclin-Cdk complexes trigger different cell-cycle events?
1. **Cyclins bind different Cdks.**
2. **The cyclin protein does not simply activate its Cdk partner** but also directs it to specific target proteins. As a result, each cyclin–Cdk complex phosphorylates a different set of substrate proteins.
3. **The same cyclin–Cdk complex** can also induce different effects at different times in the cycle, because the accessibility of some Cdk substrates changes during the cell cycle. Certain proteins that function in mitosis, for example, may become available for phosphorylation only in G2.
67
Name 3 kinases
* Cdk-activating kinases (CAKs)
* Wee1-Cdc25
* Cdk inhibitor proteins
68
What is the structural basis of Cdk activation?
Cdk-activating kinases (CAKs)
69
In CAks, is the location of the bound ATP indicated?
yes
70
The enzyme CAKs, is shown in three states. What are these states?
**(A)** In the inactive state, without cyclin bound, the active site is blocked by a region of the protein called the T-loop (red)
**(B)** The binding of cyclin causes the T-loop to move out of the active site, resulting in partial activation of the Cdk2
**(C)** Phosphorylation of Cdk2 (by CAK) at a threonine residue in the T-loop further activates the enzyme by changing the shape of the T-loop, improving the ability of the enzyme to bind its protein substrates
71
The fully active cycle-Cdk complex can be inhibited by what?
further phosphorylation at one or two sites in the active site of the enzyme
72
What does the phosphorylation of Tyr 15 by Wee1, or phosphorylation of both Thr 14 and Tyr 15 by Myt1 lead to?
The inactivation of the cyclin-Cdk complex
73
What does the Dephosphorylation by the phosphatase Cdc25 leads to?
the reactivation
74
The 3D structure of a cyclin-Cdk-CKI complex reveals what?
That CKI binding stimulates a large rearrangement in the structure of the Cdk active site, rendering it inactive
75
Why do cells use CKIs?
Primarily to help govern the activites of G1/S and S-Cdks early in the cell cycle
76
What are CKIs (Cdk inhibitor proteins)?
What is their role?
When are they active?
Proteins that interact with Cdks or Cdk-cyclin complexes to block their activity, usually during G1 or in response to inhibitory signals from the envrionment or damaged DNA
77
How can these mechanisms regulate cell cycle?
What happens after DNA damage?
→ Regulation of the cell cycle involves signal transduction via multi-step signalling pathways.
→ Cell-cycle control depends also on transcriptional regulation.
78
What is Ubiquitin (Ub)?
What occurs in its first transferral?
A small 8-KDa protein, which is first transferred ot the ubiquitin-activating enxyme, E1, in an ATP- dependent manner
79
What happens when ubiquitin is activated?
This activated ubiquitin is then transferred to the ubiquitin-conjugating enzyme, E2
80
After ubiquitin is transferred to E2, what happens to it?
Finally, the Ubiquitin is covalently attached to the target protein by E3 ubiquitin ligase, leading to the formation of a polyubiquitin chain
81
What is the polyubiquitin chain recognised by and what happens then?
The polyubiquitylated protein is recognized by the 26S proteasome and is destroyed in an ATP-dependent manner.
82
There are many E3 ubiquitin ligases, what four categories are they categorised into?
1. HECT-type
2. RING-finger-type
3. U-box-type
4. PHD-finger-type
83
What are RING-finger E3s further divided into ?
RING-finger-type E3s are further divided into subfamilies, including **cullin-based E3s**, which constitute one of the largest classes of E3s
**There are seven cullin-based E3s** including the **SKP1–CUL1–F-box-protein (SCF)** complex and the **anaphase-promoting complex/cyclosome (APC/C).**
84
Substrates of APC/C complexes are activate at what phases in the cell cycle and what activated them?
Work:
* Mid-M
* until late G1
Activators:
* Cdc20
* Cdh1
85
Substrates of SCF complexes are activate at what phases in the cell cycle and what activated them?
Work:
* Late G1
* Until early M
Activators:
* Skp2
* FBW7
* beta- TRCP
86
What does M-Cdk activity promote?
What does this result in?
* **The events of early mitosis**
This results in resulting in the metaphase alignment of sister chromatids on the spindle
* **M–Cdk activity also promotes the activation of APC/CCdc20**
which triggers anaphase and mitotic exit by stimulating the destruction of regulatory proteins, such as securin and cyclins, that govern these events
87
By promoting cyclin destruction and thus Cdk inactivation, APC/CCdc20 also triggers what?
The activation of APC/CCdh1, thereby ensuring continued APC/C activity in G1.
88
