GENE 6: Replicating the genome Flashcards
1
Q
What phase is DNA synthesised in? By what?
A
S phase
Replisome
2
Q
What does the replisome do and what enzyme does it include?
A
Includes the enzyme DNA polymerase and it adds nucleotides in the 5’ to 3’ direction to a short RNA primer
3
Q
What is the short RNA primer made by?
A
DNA primase
4
Q
What splits the DNA strand in two strands for replication
A
DNA helicase
5
Q
How many primers does the leading strand need for replication?
A
One
6
Q
How is the lagging strand replicated?
A
Synthesised from multiple primers to generate Okazaki fragments, each with its own RNA primer. Each Okazaki fragment is joined to the previous one using DNA ligament, but only after another DNA polymerase degrades the RNA primer of the older Okazaki fragment while extending the 3’ end of the new adjacent fragment
7
Q
When is polymerase alpha and delta used?
A
Alpha - lagging strand
Delta - leading strand
8
Q
What is the fidelity of DNA polymerase
A
1 error per 10^8 bp
9
Q
At what phases of the cell cycle is it important to control?
A
S and M phase
10
Q
What happens in M-phase?
A
Chromosomes segregate into daughter cells
11
Q
What three things must be ensured during the cell cycle?
A
- chromosomes are copied exactly once per S-phase
- M-phase initiates after S-phase completion
- S-phase is always preceded by M-phase
12
Q
what is aneuploidy?
A
cells with missing, extra or rearranged chromosomes
13
Q
what could failures in mitotic cells lead to?
A
apoptosis or mutations
14
Q
what could failures in meiotic cells lead to?
A
inaccuracies in chromosome duplication (meiosis I) or segregation (meiosis I and II) can generate aneuploid gametes leading to sterility, developmental defects or genetic disease
15
Q
What does meiotic cell division not undergo?
A
an intervening S phase
16
Q
How does megakaryoctye cell division differ from normal?
A
they undergo repeated S-phases with no intervening cell divisions - this leads to high enlargement and polyploidy (upto 64 sets of chromosomes) before exploding to form platelets
17
Q
what are senescent cells
A
Some cells exit the cell cycle during G1 to enter G0 which may represent a reversible resting phasewhich may represent a reversible resting phase, an extended or indefinite period of dormancy (called senescence), or the beginning of terminal cellular differentiation.
18
Q
What can senescence happen?
A
One example is when telomeres become critically short and it is no longer safe for a cell to continue dividing.
19
Q
Where does DNA replication start in circular chromosomes?
A
At the origin or replication.
20
Q
How does replication work in prokaryotes?
A
Unwinding of the chromosome at the origin allows two replication forks to form and move in opposite directions around the chromosome until the entire chromosome is duplicated
21
Q
How long does DNA replication take in prokaryotes?
A
30 minutes
22
Q
How long does replication take in mammalian cells?
A
20 hours
23
Q
How much faster is prokaryotic DNA polymerase than mammalian?
A
10x faster, 500 nucleotides per second
24
Q
How is mammalian replication different from prokaryotic replication?
A
mammalian DNA rep is initiated from multiple origins simultaneously - 1 replication origin per 70kb
Eventually the replication bubbles within each chromosome coalesce to form a fully replicated set of chromosomes.
25
How do animal replication origins differ from bacteria and simple eukaryotes?
animal cells do not have a clearly conserved DNA sequence at their replication origins
26
What is the 'landing pad' for eukaryotic assembly of proteins required for DNA synthesis to bind to called?
This complex is called the origin recognition complex (ORC)
27
What is the origin recognition complex (ORC) made up of?
composed of 6 protein subunits (ORC1 to ORC6
28
What is the first step before DNA synthesis can begin?
A pre-replicative complex (preRC) is assembled in G1, a process called origin licensing
29
What two proteins recruit replicative helicases (inactive to begin with) to the ORC?
Cdc6 and Cdt1
30
What are the helicases at the during origin licensing made up of?
Mcm proteins
31
What comes before S phase starts?
Origin activation and firing
32
How is unwanted and dangerous DNA replication avoided?
Inhibitory phosphorylation of ORC, Cdt1 and Cdc6
33
What does origin activation require?
Not only the recruitment of multiple initiation and replication proteins (inc. DNA polymerase) but also the phosphorylation of many of these proteins
34
What enzyme's activity phosphorylates the proteins needed for origin activation?
Cyclin Dependent Kinase (Cdk)
35
How do ORC, Cdt1 and Cdc6 become inactivated?
Cdk activity which phosphorylates and inactivates them (inhibitory phosphorylation)
36
How is Mcm helicase activated?
Dbf4-dependent kinase (DDK).
37
Once completion of S phase has occurred - what's next?
Entry into G2 phase
38
What's wrong with re-licensing?
it would eventually overwhelm the replication machinery - lead to DNA damage/instability/death/oncogenesis
39
How is firing no more than once per S-phase ensured?
Use of the same kinase activity both to promote origin firing and to inactivate these origin licensing components
40
When must origin relicensing be suppressed?
G2, M-phase until newly replicated chromosomes have been successfully segregated
41
What additional mechanisms have evolved to prevent unwanted origin licensing?
binding and inactivation of Cdt1 by geminin, a protein that accumulates in S and G2
42
What must a replication bubble fuse with before replication can begin? (before S-phase can be completed)
two others
43
What is different about mammalian and prokaryotic origins of DNA replication except for the number of them?
Mammalian origins of DNA usually don't have conserved DNA sequences
44
What do human origin of DNA replications determine?
Where DNA polymerase will begin DNA synthesis
45
Where do Mcm helicases load?
At or near ORC sites
46
Preventing an origin from firing more than once per S-phase requires what?
S-Cdk to inhibit origin licensing by phosphorylation of DNA helicase
47
Positive supercoils are resolved by?
Top 2 or Top 1
48
When two replicative DNA helicases encounter eachother during topological problems, what happens?
Disassembly and gap filling
49
How does DNA change from twisted to linear?
Decatenation by Top 2
50
Which are part of origin of licensing from below?
- The formation of a pre-replication complex
- The loading of MCm helicases at or near ORC sites
- The activation of Mcm by DDK
- Interactions between Cdt1 and Mcm helicase
- Inactivation of Cdct1 and Cdc6 by Cdk1
- The formation of a pre-replication complex- The loading of MCm helicases at or near ORC sites
- Interactions between Cdt1 and Mcm helicase
51
What are the two types of Cdk called and what are their purposes?
S-Cdk: promoting entry into S-phase and suppressing origin relicensing
M-Cdk: key regulator of entry into M-phase
52
What are Cdks? and what do they do?
serine-threonine protein kinases. They transfer a phosphate group from ATP onto certain serine or threonine residues in their protein substrates
53
What does S-Cdk do to Mcm and Cdt1?
S-Cdk activates Mcm helicase but inactivates Cdt1
54
What is the qualitative model?
Different cyclins accumulate at specific cell cycle stages; S-cyclins at S-phase and M-cyclins at M-phase. This suggests one way in which the same Cdk can have different effects at different cell cycle stages: by associating with different cyclins it acquires different substrate specificities at S- and M-phase. This makes the action of S-Cdk and M-Cdk qualitatively different.
55
What is the quantitative model?
The quantitative model proposes that S-Cdk and M-Cdk activities differ only quantitatively and that M-phase substrates require higher levels of Cdk activity to become phosphorylated than the S-phase substrates. As cyclin, and therefore Cdk activity, accumulates, two successive thresholds of Cdk activity are reached, the first (TS) at the G1/S boundary, the second (TM) at the G2/M boundary, to promote S- and M-phases, respectively.
56
Are the qualitative and quantitative model mutually exclusive?
No, it is possible that they are combined in different ways to generate cell cycles suited to different cell types.
57
When are cyclins degraded?
when a ubiquitin ligase complex (anaphase-promoting complex or APC/C) tags them with ubiquitin, which marks them for degradation by the cell.
58
At what stages does Cdk activity inhibit APC/C?
S, G2 and early M phase
59
At what stage does M-Cdk activate APC/C activity?
Late mitosis
60
What is the role of cohesion?
Holds sister chromatids together
61
What is the licensing inhibitor?
Geminin
62
The same signal that triggers ______ ____ _____ also ensures that cells exit mitosis into a G1 state that permits origin relicensing.
mitotic chromosome segregation
63
If Cdk activity is inhibited what may happen?
in order to cause cell cycle arrest in response to certain signals, such as DNA damage
64
Cells must first pass through _____ before they can prepare for S-phase
Mitosis
65
Why must cells first pass through mitosis before they can prepare for S-phase?
Because mitotic cyclins are degraded at anaphase
66
What condition is necessary for relicensing origins of replication?
Low Cdk activity in G1
67
Name a type of serine/tyrosine protein kinase that controls the cell cycle.
Cyclin dependent kinase (Cdk)
68
Name a type of protein that acts as an enzyme cofactor and whose concentration varies with cell cycle phase as a result of proteolytic degradation.
cyclin
69
Name a ubiquitin ligase complex activated during mitosis and responsible for degradation of cyclin, geminin and securin.
Anaphase-Promoting Complex, APC/C, APC, cyclosome
70
Name a protein heterodimer that drives cells into mitosis.
M-Cdk, M-cyclin/Cdk, Cdk/M-cyclin, M-cdk and cyclin
71
Name a challenge faced by DNA during replication
Topological stresses
72
What is required to unwind the double stranded DNA helix allow the replication machinery to access DNA?
Mcm helicase
73
Why is ATP required for unwinding the DNA
To break the hydrogen bonds between DNA strands
74
Why can the helicase not unwind the DNA indefinitely?
because the DNA ahead of the replication fork becomes overwound and topologically stressed
75
How is stressed reduced when unwinding double stranded DNA?
two types of structures accumulate: supercoils, ahead of the replication fork and, pre-catenanes (sister chromatid intertwines) behind it.
76
Why must supercoils and pre-catenanes be removed? How is this achieved?
so that replication can be completed and sister chromatids can separate. Using topoisomerases
77
What does Top1 do?
Type I topoisomerases cleave and reseal single strands in DNA supercoils, and so reduce supercoiling
78
What does Top2 do?
Type II topoisomerases cleave both strands in DNA supercoils and in precatenanes, and so can reduce supercoiling and resolve precatenanes.
79
What happens when Top2 is deleted in human cells?
Sister chromatids remain intertwined at anaphase causing delayed and abnormal chromosome segregation at anaphase and, ultimately, cell death.
80
What treatment uses Top2?
Cancer therapy as it causes double strand breaks to the DNA of cancer cells. This is possible because cancer cells are more sensitive than normal cells to DNA damage.
81
How can foodstuff potentially cause cancer? (In relation to the above)
Tow concentrations of Top2 inhibitors may be present in foodstuffs and contribute to carcinogenesis.
82
As the 5' strand is incompletely replicated, what would happen to chromosomal telomeres over time?
Chromosomal telomeres are therefore expected to become progressively shorter with successive S-phases.
83
What enzyme is used to overcome the end replication problem?
Telomerase
84
DNA polymerase can only replicate in what direction?
5' - 3' direction
85
What strand is the backstitching mechanism used in DNA replication called?
lagging strand
86
What are the two components of telomerase?
- The protein telomerase reverse transcriptase (TERT), which is 126kDa in size
- A 451 nucleotide non-coding RNA molecule (TERC) , which acts as a template for TERT to transcribe from
87
Why is there a gap at the end of the lagging strand following replication?
There is no 3'-OH group at the end of the complementary strand to prime DNA synthesis
88
How does telomerase work?
It recognises the tip of an existing repeat sequence, using an RNA template within the enzyme, telomerase replicates the parental strand in the 5' to 3' direction, and adds additional repeats as it moves down the parental strand..
89
How is the lagging strand is then completed?
By DNA polymerase alpha, which carries a DNA primase as one of it's subunits
90
What proteins are sufficient to extend TTAGGG repeats invitro?
TERT and TERC
91
What is dyskerin? What does it do?
key telomerase protein, which, along with its three accessory proteins, binds and stabilises TERC.
92
What does dyskerin binding to TERC and stabilising it ensure?
This ensures TERC is not degraded in the cell and can assemble with TERT.
93
What is shelterin composed of?
The shelterin complex comprises six proteins.
94
What does shelterin do?
Shelterin helps to protect (or 'shelter') telomeric DNA from being recognised as damaged DNA and inappropriately repaired.
95
What are telomere syndromes due to?
Mutations in the genes for many of these telomere binding proteins have been shown to underlie a range of diverse diseases
96
What occurs in telomere syndromes?
Tissues that rapidly divide tend to be most affected, as telomeres shorten rapidly without telomerase to repair them, causing cells to senesce or die.
97
Give examples of telomere syndromes
Dyskeratosis congenita
Pulmonary fibrosis
Aplastic anaemia
Hoyeraal-Hreidarsson syndrome
98
What is replicative senescence
If cells lack telomerase activity their telomeres shorten with increasing cell divisions until a program of cell senescence is activated.
99
What is the Hayflick limit and when does it occur?
After about 50 doublings (known as the Hayflick limit, after its discoverer) they stop dividing and enter a senescent state.
100
What happens when too high levels of telomerase are produced in cells?
predisposition to oncogensis
101
When are high levels of telomerase needed in cells?
in cells that need to undergo extensive proliferation, including germ cells, early embryo cells and somatic stem cells. Telomerase expression in these cells allows telomere lengths to be maintained or extended in order to resist senescence.
102
When telomeres are not well protected by shelterin, what can happen?
If TRF2 is mutated, chromosome ends can fuse with one another or to broken DNA. Such events are highly genome destabilising, mutagenic and therefore potentially oncogenic.
103
What is the sequence of repeats that Telomerase adds to the end of the lagging strand?
TTAGGG
104
Name a protein complex that prevents telomeric DNA being recognised
Shelterin
105
What is the subunit of telomerase with reverse transcriptase activity?
TERT
106
What is a component of telomerase that acts as a reverse transcription template?
TERC
107
What is a component of shelterin is implicated in preventing telomere fusion?
TRF2
108
Concerning Topoisomerase II (select all correct statements):
1) It is essential for cell viability
2) It is the only way to relax positive supercoils.
3) It cuts both strands of the DNA double helix
4) It a target for anti-cancer drugs.
5) Is necessary for the initiation of DNA replication
1) It is essential for cell viability
3) It cuts both strands of the DNA double helix
4) It a target for anti-cancer drugs
109
Select all correct statements.
1) Excessive telomerase activity increases the risk of oncogenesis.
2) Impaired telomere maintenance increases the risk of oncogenesis.
3) Up-regulating telomerase is an acceptable approach to increasing human life-span.
4) Inhibiting telomerase in cancer cells is an acceptable approach in anti-cancer research.
5) Many telomere syndrome symptoms can be explained by loss of somatic stem cells.
1) Excessive telomerase activity increases the risk of oncogenesis.
2) Impaired telomere maintenance increases the risk of oncogenesis.
4) Inhibiting telomerase in cancer cells is an acceptable approach in anti-cancer research.
5) Many telomere syndrome symptoms can be explained by loss of somatic stem cells.
