Lecture 6

Question 1

Why do cells work so hard to preserve their DNA integrity?

Answer 1

DNA is the carrier of genetic information
Changes in the DNA, such as mutations or chromosomal rearrangements, can result in changes in gene activity, leading to: impair cell function or cancer/cell transformation

Question 2

What are the different type of DNA dammage?

Answer 2

1. Mismatch of bases: when a wrong base is inserted into DNA during replication. (both strands look normal so info could be lost about which is the original strand —> done based on presence of DNA methylation)
2. Change to a single base: Oxidation (react with oxygen), Hydrolysis (react with water)
3. Base loss: formation of an abasic site
4. Complex modifications involving multiple base pairs and inter-strand crosslinks: such as UV-induced pyrimidine dimers (adjacent pyrimidine)
5. DNA breaks, including single-strand or double-strand breaks.

More complex forms of DNA damage can be created when a replication fork (DNA polymerase) or a transcription bubble (RNA polymerase) collide with damaged DNA

This (and other factors) lead to the general principle that metabolically active and rapidly proliferating cells don’t tolerate DNA damage as well as quiescent cells (active cells are more sensitive to DNA damage, used in cancer therapy)

Question 3

What are the main sources of DNA dammage?

Answer 3

EXOGENOUS (ENVIRONMENTAL)
1) Radiation: UV light, X-rays, γ-rays
2) Chemical Mutagens: cigarette smoke, environmental pollution, various drugs
ENDOGENOUS (PHYSIOLOGICAL)
4) Replication errors
5) Spontaneous chemical reactions: Hydrolysis, Oxidation
6) Mutagens formed by our metabolism: reactive oxygen species (ROS) produced by mitochondriae and immune cells
*Spontaneous mutation rate during nuclear DNA replication is very low – less than 1 in 10^9 bases per cell division

Question 4

What are the major DNA repair pathways?

Answer 4

1. Base excision repair —> Single damaged nucleotide
2. Nucleotide excision repair —> Complex damage affecting multiple nucleotides
   Can remove various complex helix- distorting modifications affecting multiple base-pairs. A distorted region is recognized, and two incisions are made on either side to excise the damaged DNA strand. The resulting nucleotide gap is filled by DNA polymerases.
3. Mismatch repair —> Mismatched Bases
   Removes mismatched bases. It is initiated by mismatch recognition proteins, and a single-strand segment of DNA is excised between the mismatch and a nearby nick. The gap is filled by specialized DNA polymerases.

Question 5

How does the base excision repair pathway work? What does it deal with?

Answer 5

Base excision repair (BER) typically mediates the removal and replacement of a single damaged residue.
Substrates include uracil residues in DNA and damaged bases ( e.g. base oxidation, hydrolysis).
- Damaged base is removed by a DNA glycosylase (e.g. UNG for uracil), backbone stays intact
- Backbone at the resulting abasic site is excised by APEX1 endonuclease.
- One nucleotide gap is filled by DNA polymerase β.
- DNA joining is catalyzed by DNA Ligase III. (bond in the back bone)

Question 6

How do our cells repair double strand DNA breaks?

Answer 6

Non-homologous end joining:
Uses local DNA at site of break, can act at any phase of the cell cycle but potentially mutagenic
CISPR-Cas9 uses this idea that NHEJ is slightly mutagenic
- Dangerous if a cell has multiple DNA breaks as 2 ends not supposed to be connected get rejoined

Homologous end joining:
HR only operates when a copy of the damaged DNA sequence is available, usually as a sister chromatid in G2 phase of the cell cycle.
Search for DNA information in the sister chromatid
Not mutagenic, but restricted to G2 phase of cell cycle

Question 7

What are the steps of NHEJ?

Answer 7

Non-Homologous End Joining (DNA repair)
Step 1. Recognition of broken DNA ends by Ku70/Ku80 protein heterodimers.
Step 2. Recruitment and activation of DNA-dependent protein kinase catalytic subunit (DNA-PKcs)
Step 3. DNA rejoining by DNA Ligase IV in complex with XRCC4.
Various auxiliary factors are also involved: ATM, Artemis, NBS1- MRE11-RAD50. (no need to memorize these)

Question 8

What is a classical Role of Chromatin in the recruitment of Repair Machinery (DNA damage)?

Answer 8

Phosphorylation of histone variant H2AX:
By kinase ATM is one of the first steps in DNA damage response. phospho-H2AX is also known as γH2AX and accumulates at DNA damage foci. It can be easily detected by immunofluorescence microscopy in irradiated cells.

Histone H2AX is a variant of H2A incorporated into DNA when DNA damage
When DNA break, one of the 1st events is phosphorylation of Histone H2AX by kinase ATM.
Could count DNA breaks in the nucleus by doing immunofluorescence microscopy for yH2AX (phosphorylated)

• Phospho-H2AX acts as a binding site for protein MDC1 and recruits it to DNA damage foci.
• MDC1 is then phosphorylated by kinase ATM and recruits ubiquitin ligases RNF8 and RNF168.
• RNFs ubiquitinate histone H2A and many other proteins at the DNA damage foci.
• Polyubiquitin chains then recruit many further DNA damage response and repair factors.

Question 9

How does the cell choose between HR and NHEJ repair pathways?

Answer 9

• Recruitment of 53BP1 favors NHEJ
• Recruitment of BRCA1 favors HR.

Question 10

How does the cell choose between HR and NHEJ repair pathways?

Answer 10

• Recruitment of 53BP1 favors NHEJ
• Recruitment of BRCA1 favors HR.

Question 11

What are different responses of the cell to DNA damage?

Answer 11

1. Cell cycle checkpoint activation
2. Transcriptional program activation
3. DNA repair mechanisms
4. Apoptosis

Question 12

What is the MAIN protein involved in DNA damage?

Answer 12

p53 → Tumor suppressor & Transcription factor

Question 13

What type of protein is p53? What about its structure?

Answer 13

1. Tumor suppressor
2. Transcription factor
   Master regulator of cellular responses to DNA damage and many other types of cellular stress
   N terminal → transcriptional Activation Domain, site of binding of inhibitor MDM2
   Core → Sequence-specific DNA-binding domain
   T domain → Tetramerization domain
   C terminal → Non-specific DNA binding domain
   *It is the most frequently mutated gene in human cancers!

Question 14

How does MDM2 regulate p53?

Answer 14

MDM2 is the master regulator (inhibitor) of p53 stability - it catalyzes polyubiquitination of p53, targeting it to proteasome for degradation.
In turn p53 stimulates MDM2 gene expression, creating a self-regulating negative feedback loop.
- In healthy cells, p53 is quickly degraded (levels are kept very low)

Question 15

How is p53 activated in response to DNA damage?

Answer 15

DNA damage activates ATM and other kinases, that phosphorylate both p53 and MDM2 to disrupt their interaction. This results in stabilization and activation of p53.

Question 16

What are cellular responses induced by activation of p53?

Answer 16

Cellular responses to p53:
- Cell cycle arrest (p21)
- Apoptosis (PUMA, NOXA)

Question 17

How Does p53 Induce Cell Cycle Arrest?

Answer 17

• Cell cycle progression is regulated by cyclin-dependent kinases (CDKs), with different CDKs active at different phases of the cell cycle.
• CDKs are activated by the binding of small regulatory proteins cyclins, and inhibited by CDK-inhibitors (CKIs).
• Expression of both cyclins and CDK- inhibitors is strictly regulated.
• p21 (also known as CDKN1A, cyclin-dependent kinase inhibitor) is an inhibitor of CDKs 2,4,6. Induction of its expression by p53 therefore leads to cell cycle arrest. (inhibits kinases active in G0 and G1 phases)
  *P53 induces activation of p21 which in turn arrests cell cycle

Question 18

What is cell senescence? How is it induced?

Answer 18

Irreversible cell cycle arrest
1. CDK-inhibitors involved: p16 (also known as CDKN2A) inhibits CDKs4/6-CyclinD in G0/G1 phase of cell cycle
2. Connection to P53: p19 is another protein expressed from the same locus as p16. Its role is to bind and repress MDM2 activity, leading to p53-stabilization.
*positive feedback loo where 053 activates p16 → which … → further stabilizes p53

Question 19

How does p53 mediated induction of apoptosis occur?

Answer 19

*cell examines its own internal state and choses apoptosis faith (cell intrinsic in the context of damage)
This is the so-called ‘intrinsic’ pathway of apoptosis:
Checkpoint = integrity of outer membrane of mitochondria
1. Apoptosis induction involves the release of mitochondrial protein cytochrome C into the cytosol
2. Cytochrome C assembles with APAF1 and procaspase 9 into the apoptosome complex.
3. This leads to the activation of caspase 9, caspase 3, etc.

Integrity of Outer Mitochondiral Membrane (OMM) is maintained by BCL2 Family of Proteins – regulators of the Mitochondrial Membrane Stability
- Can be apoptotic or anti-apoptotic

Question 20

What are 2 pro-apoptotic BCL2 family members?

Answer 20

BCL2 Family of Proteins – regulators of the Mitochondrial Membrane Stability
NOXA & PUMA
p53 activates the expression of proapoptotic members of the BCL2 family,
such as PUMA and NOXA.
This shifts the balance of BCL2-protein activity towards mitochondrial membrane permealization, cytochrome c release, and apoptosis

Question 21

What are the general steps of a ChIP-Seq protocol?

Answer 21

1. Crosslinking of protein to DNA with paraformaldehyde.
2. Fragmentation of DNA into 150-300 bp fragments.
3. Immunoprecipitation of the transcription factor of interest using an antibody. crosslinked DNA will be pooled down as well.
4. Reversal of crosslinks, DNA isolation, and analysis either by sequencing or by quantitative-PCR.
   With Chip-Seq all regions of the genome bound by a transcription factor of interest can be identified.
   *This and other analyses reveal that p53 acts as a transcriptional regulator for hundreds of genes!

Question 22

Using ChIP-Seq we were able to identify novel p53-target genes and functions under both stress and homeostatic conditions. Which are they?

Answer 22

p53 also regulates:
- Antioxidant response
- Metabolic activity, including the induction of autophagy
- Cell secretome and migratory activities
- Stem cell differentiation overself-renewal

Question 23

What is the specific role of p53 in Stem Cells?

Answer 23

Can p53 act a transcriptional repressor (as well as activator)? p53 is reported to repress the expression of the key ES-cell pluripotency transcription factor NANOG + activate expression of differentiation-linked genes (hence why stressed cells are hard to maintain stemness)
Other pluripotency genes are repressed through p53-mediated induction of microRNAs such as mir34A and mir145.
*Reprogramming protocoles are difficult because of p53 activation (reprogramming can be seen as carcinogenic), much easier to reprogram p53 KO cells

Question 24

Why is DNA Damage in Stem Cells Especially Dangerous?

Answer 24

• Tissue stem cells have to persist throughout the lifetime of an organism and differentiate to contribute to the production of many downstream differentiated cells of a tissue.
• Therefore, the consequences of DNA damage in stem cells are potentially a lot more dangerous than the consequences of DNA damage in terminally-differentiated cells.
• Unlike terminally-differentiated cells, tissue stem cells are already capable of indefinite self-renewal. Therefore, the number of genetic changes needed to transform them into cancer stem cells is lower than that for terminally- differentiated cells. 

  *So, are there any stem-cell specific mechanisms for DNA damage response and repair?

Question 25

Do stem cells have specific adaptations that minimize their exposure to physiological stress, mutagens, and DNA damage?

Answer 25

YES - Efflux of Cytotoxic Dyes by Stem Cells - Stem Cell Quiescence (when divide, rarely, generate progenitor cells and these progrenitors will undergo crazy proliferation and differentiation, but the stem cells will not) - Hypoxic Environments of the Stem Cell Niches 


Question 26

What are observations/facts about Efflux of Cytotoxic Dyes by Stem Cells?

Answer 26

Stem cells strongly efflux certain cell- penetrating dyes e.g. Hoechst33342 or Rhodamine 123, due to their high expression of the ATP-binding cassette (ABC) transporter family proteins. This is the basis of the traditional approach of enriching stem cells as the ‘side-population’, which is negative for the staining with these dyes, by flow cytometry. Is this also an adaptation to minimize DNA damage?

Question 27

How can we assess Stem Cell Quiescence?

Answer 27

Hematopoietic stem cells (and many other somatic stem cells) are largely quiescent and therefore in the G0-phase of the cell cycle.
 Quiescence is one of the mechanisms protecting stem cells from excessive physiological stress, as compared with the more mature rapidly-dividing progenitor cell populations.
- protection from mistakes in DNA replication,
- low energy demands mean low metabolic rate and low reactive oxygen species levels

Question 28

How can we identify G1 vs G0 cells?

Answer 28

Both G1 & G0 have lower DNA content, but G1 has higher RNA content as compared to G0

Question 29

What does the quiescent state of stem cells allow, which helps with reduction physiological stress?

Answer 29

Quiescent state means low energy demands Stem cells rely on non-oxidative glycolytic metabolism Stem cells have low mitochondrial content This means that stem cells can reside in low oxygen environments in our tissues Altogether, this leads to low levels of mutagenic reactive oxygen species **Hypoxic Environments of the Stem Cell Niches

Question 30

How can we observe that Stem cells actually prefer hypoxic envrionments?

Answer 30

Hematopoietic stem cells are critically dependent on the HIF-1α hypoxia signaling pathway. Modulation of this pathway can enhance or impair stem cell function. In healthy cells, HIF-1a levels are kept very low, but in hypoxia, HIF-1a accumulates. If delete HIF-1a in HSCs, associated with loss of quiescence, low stress resistance, increased mtROS Deletion of VHL (which degrades HIF-1a) is associated with enhanced quiescence, higher BM chimerism, reduced mtROS

Question 31

What is an importance specialized Mechanisms for DNA Maintenance and Repair in Stem Cells?

Answer 31

Telomere maintenance in Stem Cells

Question 32

What are telomeres?

Answer 32

Telomeres are the structures found at the end of our chromosomes They consist of non-coding repetitive DNA, and are associated with the Shelterin protein complex that protects the telomeres Some cells, not all, can’t express Telomerase complexe which maintains telomere length (if not, telomeres shorten with each DNA replication process) - hTERT = protein component of Telomerase Complex - hTERC = RNA component of Telomerase Complex *5’ end always shorter than 3’ end 3’ ends of the DNA strand can’t be effectively replicated, because: 1. an RNA-primer is used to initiate the synthesis of a new DNA-strand 2. once the RNA primer is lost, the 5’ end of the newly synthesized strand is shorter than the original template.

Question 33

How is telomere length maintained?

Answer 33

Some cells, not all, can’t express Telomerase complexe which maintains telomere length (if not, telomeres shorten with each DNA replication process) - hTERT = protein component of Telomerase Complex - hTERC = RNA component of Telomerase Complex

Question 34

What is the result of Telomere shortening?

Answer 34

Telomeres get shorter in most cells in our body as we age - Short unprotected telomeres are dangerous and get sensed by the cell as DNA-damage - Telomere shortening triggers p53- dependent stress response and induces cellular senescence. - If it doesn’t, unprotected telomeres result in chromosome translocations and fusions 
*Some cancer cell lines express telomerase do counter-act this or downregulate p53

Question 35

What is Telomerase?

Answer 35

Telomerase is an enzyme complex that extends the 3’ ends of telomeric DNA Expressed in stem cells and cancer cells Consists of the protein component TERT and RNA component TERC - Germline cells express high levels of telomerase - Tissue stem cells express it, but not quite as high (still some telomere shortening, but slower)

Question 36

How do normal DNA repair pathways affect Tissue Stem Cell Maintenance?

Answer 36

1. In human, congenital defects in various DNA repair pathways are also linked to bone marrow failure diseases – resulting from hematopoietic stem cell depletion - Homolgous recombination Nucleotide excision repair ~ Fanconi anemia - NHEJ ~ Ligase IV syndrome - Telomere maintenance ~ Dyskeratosis congenita (TERT, TERC) - Multiple DSB-repair pathways ~ Ataxia telangiectasia (ATM) 2. Defects in other DNA repair pathways affect other rapidly-renewing tissues: - Nucleotide excision repair ~ Xeroderma pigmentosum (with skin cancer) - Mismatch repair ~ Increased risk of colorectal cancer

Question 37

Which pathway is used/prefered to repair DNA Breaks in Hematopoietic Stem Cells?

Answer 37

Non-homologous end joining: - In G0/G1 phases of cell cycle - more error-prone than HR Homologous recombination: - Restricted & preferred in G2 phase of cell cycle - Tries to recover information from sister chromatid *Example of how Stem Cells might be at a disadvantage compared to other cells because restricted to a less efficient pathway in G0/G1 in which they spend a lot of time

Question 38

What gives Tissue Stem Cells a specially protected status compared to other cells? (at the DNA level)

Answer 38

1. Stem cells having specific adaptation that minimize their exposure to physiological stress, mutagens, and DNA damage 2. Specialized mechanisms for DNA maintenance and repair 3. Specialized Regulation of DNA Damage Response in Tissue Stem Cells 4. Stem cells play a role in DNA damage and DNA damage response

Question 39

Why is their a need for balancing the DNA damage response in Tissue Stem Cells?

Answer 39

DNA replication errors, metabolism/oxidative stress, external mutagen → DNA damage in Tissue Stem Cells 2 possible outcomes: 1) Tolerance of DNA damage or 'mutagenic' repair → Genomic Instability → Cancer 2) Stringent DNA Damage response → Cell death or Senescence → Tissue degeneration / Ageing

Question 40

How does specialized Regulation of DNA Damage Response in Tissue Stem Cells confers them an advantage?

Answer 40

Stem cells are less prone to p53-induced apoptosis than progenitor cells: - Quiescent hematopoietic stem cells (HSCs) are predisposed to survive DNA damage induced by low-level radiation through a DNA repair process called non-homologous end joining (NHEJ), which tends to be error prone. *Survival - In contrast, progenitor cells progressing through the cell cycle are more likely to undergo apoptosis or repair their DNA using higher-fidelity homologous recombination (HR). - Although the short-term consequence of HSC survival is the maintenance of tissue integrity in the face of injury, long-term consequences are mutations and genomic rearrangements persisting in HSCs with a diminished functional capacity.

Question 41

What is Clonal Hematopoiesis?

Answer 41

It is a condition that occurs in elderly individuals. - Progressively lower number of HSCs sustain the hematopoietic system as we age - Many of the persisting active HSCs accumulated mutations that give them a competitive advantage over wild type HSCs - Such mutations are commonly in genes encoding epigenetic regulators, splicing proteins, and tumor DNA damage response pathways *Very common, so not considered a disease on its own, but increases risks of MDS (Myelodysplastic syndrom), which is a pre-malignant state and which increases risk of AML (Acute Myeloid Leukemia)

Question 42

What are the Roles of Stem Cell DNA Damage and DNA Damage Response in Ageing? What are different perspectives on the causes and mechanisms of ageing? (Is DNA damage accumulation in tissue stem cells the driving mechanism of tissue ageing?)

Answer 42

- Ageing is a STOCHASTIC process, caused by unavoidable “wear-and-tear”... things just go wrong with time... - Natural-selection argument: evolution does not eliminate deleterious mutations if their effects are only seen beyond the reproductive years. In fact, natural- selection may favor genetic changes that make us “fit” during reproductive years even if they impair us in the old age. - Molecular basis of ageing: are the same mechanisms that protect us against cancer responsible for ageing? For example, p53- mediated response to DNA damage or telomere attrition leading to cell senescence and apoptosis. (Not about the acquisition of mutations, more about how we respond to these) - Stem cell cellular basis of ageing: Are the ageing phenotypes we see at the tissue- level primarily caused by impaired function in the stem cell compartment of the ageing tissue? (loss of capacity to regenerate the tissue)

Question 43

How does Ageing affect Hematopoietic Stem Cells?

Answer 43

- Aged HSCs have decreased regenerative capacity, particularly under stress. - Aged HSCs have an altered differentiation program: decrease in production of common lymphoid progenitor and consequently B and T cells, with increased production of granulocyte–macrophage progenitors (GMPs) and consequently granulocytes and macrophages. (Biased towards production of myeloid lineage cells, decrease production of Erythrocytes) - Myelodyspastic syndrome is a common disorder of hematopoiesis in the elderly, and can progress to myelogenous leukemia (AML).

Question 44

Can p53- mediated DNA Damage Response Affect Ageing?

Answer 44

Evidence for the Ageing Roles of P53: - Mouse lines expressing a mutant hyperactivated p53 show symptoms of premature ageing, but are significantly resistant to cancer. - With age, tissues derived from these mice develop increased numbers of senescent cells, and their HSCs are functionally impaired. 
 Evidence for Role of p53 in Longevity: - But transgenic mice carrying one extra copy of an intact (wild-type) p53 gene have an up to ~15% longer lifespan, with cancer resistance.

Question 45

What are Novel functions of p53 under homeostatic conditions which promote repair without causing cell death or senescence ?

Answer 45

p53 also regulates: - Antioxidant response - Metabolic activity, including the induction of autophagy - Cell secretome and migratory activities - Stem cell differentiation over self-renewal

Question 46

What are different applications of studying DNA Damage Response in HSCs?

Answer 46

1) Developing enhanced protocols for the targeting of cancer stem cells Concept of Cancer Stem Cells : - Tumors are not homogeneous but hierarchical. Only a small proportion of tumor cells is capable of indefinite self- renewal and therefore responsible for long-term propagation of the tumor. - These are the cancer stem cells. Elimination of cancer stem cells is key to curing cancer. - These concepts apply very well to some (AML) but not so well to other cancers (melanoma). 2) Understanding DNA damage response to enhance CRISPR/Cas9 gene editing - « CRISPR-Cas9 genome editing induces a p53-mediated DNA damage response »