AMC-HC 5+6: DSB repair & Immortalization Flashcards
Histone octamer
2 times: H2A, H2B, H3 and H4
DNA damage causes
-Extrinsic factors
-Metabolic pathways
-Replication
At DSB’s, certain proteins are visualised as ‘foci’. Which proteins are these, and why are they visible even in normal conditions?
Proteins involved in DNA repair. There are always visual due to background DSB.
When are the foci of DNA repair proteins concentrated around certain parts of the DNA?
When DSB’s occur due to e.g. Ionizing radiation > relocate to the site of DNA damage.
Risk of DSB
1 double strand break could lead to an 1-2000 bp modification and LOH
How do repair proteins relocate to the DNA damage?
At random by diffusion: they bump into the DNA damage and get stuck there.
Laser microirradiation could be used to quantify the accumulation of DNA repair proteins around DSB’s. What are the problems with the technique?
-The dose is difficult to control
-Mostly UV-light DNA lesions
-Large dose is concentrated in a small area of the nucleus.
Features of Ultra-soft X-ray irradiation microscope
-Exactly calculate the dose
-Clinically relevant radiation type
-Spatial control over the irradiation patterns > metal plates with holes or stripes
-Real-time imaging
DSB repair signaling
DSB detection by ATM/MRN complex and DNA-PK
> ATM autophosphorylates itself and it phosphorylates a certain histone.
> Transduction: ATM phosphorylates Mdc1 > activates UBC13-RNF8.
> RNF8 is replaced by RNF168 (UBC13-RNF168)
> Activation 53BP1 and dimerization > decides which pathway to use for specific DSB
Onset of accumulation at individual DSBs is ….
Asynchronous: some proteins arrive very late (DSBs are relatively dangerous)
Bad timing of RNF8 and RNF168
They arrive earlier than Mre (onderdeel MRN complex), Mdc1 and 53BP1
Is the duration of the accumulation of DSB signaling proteins consistent?
No, its variable (even within the same cell nucleus)
Is distance between the nearest foci correlated with time of arrival at the DSB?
No
Which DSB signaling protein arrives way faster than the others at a DSB?
RNF168
Histone acetylation affects the ….
chromatin organisation
Histone acetyl transferases (HATs)
Add acetyl group onto histones > chromatin relaxation
Histone deacetylases (HDACs)
Remove acetyl groups > chromatin condensation
Effect of HATs on RNF168 accumulation inititation
Histone acetylation affects accumulation chromatin (creation euchromatin) > faster accumulation
DNA condensers like DRAQ5 have a …. on time of arrival of RNF168 to the DSB
negative (slowing)
DSB signaling kinase (ATM or DNA-PK) inhibition (by inhibitors) …. accumulation of RNF168
Delays
Alpha particle radiation
-Accelerated helium ions (alpha particles) are known to induce complex DNA lesions
- Does accumulation kinetics depend on lesion complexity?
-Creating linear track of DSB lesions caused by one alpha particle: more complex DSBs
Conclusion of Alpha particle radiation
DSB complexity impacts accumulation onset and duration
> acceleration of onset with high complex DSBs
Free-radical involvement: how are cells exposed to ionizing radiation induced to DSBs
-In cells exposed to ionizing radiation, a large fraction of DNA DSBs are induced indirectly via oxygen radicals formed due to water radiolysis
Oxygen radical danger
Oxygen radicals are short-lived but extremely reactive and can cause DNA damage upon contact
DMSO
A known free-radical scavenger
Directly- and radical- induced lesions differ in complexity: true or false
True
Effect of free radical removal on accumulation onset of RNF168
Delay
Effect of free radical removal on duration of RNF168
Non
Normal cells can only replicate a small number of times. What is serial passaging used for?
Determine the maximum number of divisions in vitro
What happens after exceeding division maximum?
Cease proliferation or apoptosis
What do the normal cells enter after the replication limit?
Senescence (in culture)
- Remain metabolically active
- Loose the ability to re-enter the cell cycle
- Spread out in monolayer culture
- Acquire a large cytoplasm
- Persist for weeks/months
- Fried egg appearance (microscope)
Where does the replicative doubling limit depend on?
-Species
-Tissue of origin
- Age of donor organism
Embryonic stem cells are ….potent
pluripotent, can generate all other lineages
ES cells in culture are …
Immortal: unlimited replicative potential
Immortality is a feature of single cells; true or false
False, it’s of the cell lineage
Cancer cells resemble …
ES cells, can replicate indefinitely in culture
Which cultured cells can also replicate indefinitely in culture?
HeLa cells
Why do cancer cells need immortality for transformation?
Limited division potential could be a anti-cancer strategy
> 40 cell doublings needed for tumorigenesis assuming exponential growth
> some normal cells can divide over 50 times in vitro
Some lineages of colon carcinomas extend over 2000 cell generations. What does this learn us?
Tumor growth is not exponential
Systems that cells limitate their dividing potential with
-Accumulation of physiological stress
-Recording the number of cell divisions
Senescence is accompanied by increasing expression of two CDK inhibitors. Name them and their function
p16 and p21 > halt the cell cycle
(also p53)
Which CDK inhibitor is often mutated in cancer?
p16
Function of CDKs
Control the cell proliferation
Reduction of culture oxygen leads to …. in vitro
Increased replicative life span
Why does reduction of culture oxygen lead to increased replicative life span
The high oxygen culture causes progressive breakdown of mitochondrial function and accumulation of ROS > oxidative damage > induction senescence
Culturing cells in unsuitable medium like plastic leads to more … and thus a …. in replicative lifespan
More p16, decrease replicative lifespan
Ectopic expression
Expression of the gene at a location where the product does not function
How can senescence of human cells be eliminated?
Ectopic expression of SV40 large T antigen > Large T inactivates pRb and p53, which both control cell survival pathways
> stimulating proliferation and replicative life span
Ki67 is a … marker
Proliferation
Triggers of senescence in vivo
-Physiological stress
-Hyperoxia
-DNA damage created by ROS, X-rays, or chemotherapeutic drugs.
Are there special growth media which can prolong cell replicative lifespan?
Yes
Senescent cells can also stimulate tumurigenesis. How?
Release of pro-inflammatory cytokines.
Can SV40 Large T oncoprotein immortalize human embryonic kidney cells?
No, it can only bypass senescence.
What happens to linear DNA if you transfect cells with them?
The transfected linear DNA molecules are rapidly fused end-to end by the repair mechanism > recognition as break points
- Defense mechanism against foreign DNA of for example viruses (transfection is mostly performed with circular DNA).
Cells are able to enter a state of crisis, characterised by what?
Widespread apoptosis and genomic instability
Telomeres are the protection mechanism of linear DNA chromosomes from end-to-end fusion of the chromosomes. How?
Forming of T-loops (and D-loops)
What happens to chromosomes if they were to lack telomeres?
They would fuse to each other.
Repeat sequence of the telomere
TTAGGG
What happens to telomeric DNA of proliferating cells?
They shorten during each division cycle (RNA primer disappears > polymerase needs free 3’- end)
How do telomeres appear in cancer?
Short but stable
What happens with cells with short telomeres?
With short telomeres, the cell enters a state of crisis > fusion of chromosomes (the short chromosomes cannot protect the ends of the chromosomes) > apoptosis
Breakage-fusion-bridge cycle
During anaphase the bridge created between two joined homologous chromosomes get broken because of the pulling microtubules from the centrosomes. Another nonhomologous chromosome fragment rejoins the broken chromosome and since these breakpoints are random, the chromosomes gets all mixed up with different lengths.
How are telomeres formed
Repeat hexanucleotide sequence TTAGGG
-One of the strands is longer and froms the T-loop around the shorter strand. But to exactly cover the loop of the short strand, a loose loop of the long strand hangs out because the strands aren’t the same length. This is the D-loop (displacement loop)
Function of T-loop
Protection against DNA degradation or DNA repair
T-loops are bound by ….. (name and function)
Shelterin complexes, many copies on the repeats help forming the T-loop.
At what position of the telomere, is the DNA 3-stranded?
A the D-loop
The end-replication problem isn’t the largest contribution to telomere erosion. What is?
Exonucleases within cells which chew on the ends of telomeric DNA.
How do telomeres protect the cell against transformation?
When telomeres get too short and there is potential for DNA damage due to erosion, the breakage-fusion-bridge cycle is initiated and this leads to crysis > apoptosis
How can cancer cells escape crisis?
Expressing high levels of telomerase.
Function of telomerase
Extending the telomere length > telomerase is a reverse transcriptase which contains its own RNA primer with telomere repeat antisense sequence
Natural telomerase expression
Strongly expressed during embryogenesis and later at low levels.
Catalytic subunit of telomerase (human)?
hTERT: human telomerase reverse transcriptase.
How are some cells able to maintain telomeres without telomerase?
The telomerase-independent ALT mechanism
- Interchromosomal copying mechanism
- Polymerases replicating DNA on one chromosome may use sequences from a second chromosome as a template before switching back.
> then the 5’ end strand gets extended.
Name another mechanism for interchromosomal DNA transfer
Uneven crossing-over between 2 chromosomes
> extension of telomere of A and shorter telomere of B.
Are there DNA repair proteins involved in the ALT pathway?
Yes, some are
Mouse vs human telomeres
-Mouse telomeres are 5x longer (telomere shortening doesn’t inhibit tumorigenesis)
-Expression of hTERT is differentially controlled > hTERT k.o. mice are normal up to 5th generation.
-Mouse cells > easily immortalized.
-Mouse: shorter lifespan
Does a relationship between aging and cancer susceptibility exist with regard to telomere erosion?
Telomere length does decrease with age, but there is a high variability between individuals, tissues, cells and chromosomes
> other processes play a role as well in aging
Telomere-induced foci
Disfunctional telomeres are recognised as DSBs and will attract repair proteins > creation of telomere-induced foci of repair proteins
