Teodoro Lectures

Question 1

What is the end replication problem?

Answer 1

The end replication problem arises during DNA replication, particularly affecting linear DNA.

It occurs because DNA polymerases cannot fully replicate the ends of linear chromosomes.

Without mechanisms to help with this problem, DNA strands would shorten with every replication, leading to potential loss of essential genetic information.

Question 2

What is the mechanism of DNA replication?

Answer 2

1) Leading strand: it is synthesized continuously. The DNA polymerase moves processively along the template strand until it reaches the end, completing replication without leaving gaps. (no issue)

2) Lagging strand: it is synthesized discontinously, RNA primers initiate synthesis of short fragments (okazaki fragments), after RNA primer removal, gaps remain where the primer was located, leading to incomplete replication of the chromosome end, this results in progressively short DNA strands after each replication cycle (issue ** end replication problem**)

Question 3

What are coping mechanisms in organisms for the end replication problem?

Answer 3

1) Circular Genomes:
- bacteria and viruses, circular genomes do not have ends, so this completely avoids the end replication issue.

2) Linear Genomes:
- Telomeres:
-> found at the ends of linear chromosomes in eukaryotes, composed of repetitive non-coding sequences (telomeric repeats), these sequences are added by the enzyme telomerase, which helps maintain chromosome length during replication.

Question 4

How do Small DNA viruses deal with the end replication problem?

Answer 4

• many small viruses (some causing cervical cancer) have circular genomes, allowing them to avoid to issue.

Question 5

How does Large DNA viruse: Pox Virus, deal with the end replication problem?

Answer 5

1) Pox virus:

• structure: membrane-bound core containing a long linear genome (around 200,000 base pairs)
• mechanism: the virus employs unique enzymes that covalently seal the ends of the DNA to stabilize it.
• Terminal Groups: Seal the ends of the DNA, preventing degradation.
• Tandem Repeats: Allow for complementarity at the ends, enhancing stability.
• AT-Rich Regions: Designed with mismatches that create weak base pairing, facilitating melting during replication.
• self-priming mechanism: the terminal repeats can fold back and anneal, allowing virus to utilize leading strand synthesis, enabling the viral polymerase to replicate the DNA effectively.

Question 6

How does Large DNA viruse: Adeno Virus, deal with the end replication problem?

Answer 6

• Structure: linear double-stranded genome (40,000 base pairs, containing a terminal protein (TT protein) covalently attached to the 5’ end of the DNA.
• Replication mechanism:
  Inverted Terminal Repeats (ITR): Present at both ends of the adenoviral genome, comprising 100 nucleotide repeats. This terminal protein facilitates the priming of DNA replication right at the ends.
• the terminal protein recruits’ viral DNA polymerase, ensuring precise positioning for replication. Other proteins, such as single-stranded DNA binding proteins, assist in stabilizing displaced strands during the replication process.
• newly synthesized viral genome and displaced viral genome can fold back to form a panhandle structure, enabling efficient replication.

Question 7

Telomerase In Eukaryotes/mammalian cells

Answer 7

• enzyme that adds repetitive nucleotide sequences to the 3’ ends of linear chromosomes.
• this extension compensates for the inability of DNA polymerase to fully replicate the ends of linear DNA.

Question 8

How do insects (e.g., Drosophila) deal with the end replication problem?

Answer 8

• do not encode telomeras enzyme
• utilize retrotransposons to maintain the ends of their chromosomes

Question 9

What are the types of transposons? What is the mechanism that takes place?

Answer 9

1) Healing transposon
2) Telomere-associated transposon 3)Het-A related transposons

• Drosophila has HTT arrays (HeT-A, TART, Telo-Associated Retrotransposons) at the end of their chromosomes.
  -> the reigions containing HTT arrays are transcribed into mRNA, the mRNA is polyadenylated and transported to the cytoplasm, mRNA forms with a ribonucleoprotein complex with reverse transcriptase, this returns to the nucleus and uses the mRNA as a template to synthesize DNA.
  -> this process involves target-primed reverse transcription: RNA serves as a template for synthesizing additional telomeric DNA copies, adds more telomeric sequences to the ends of the chromosomes, compensating for any potential loss during replication.

Question 10

Why do we need Telomeres?

Answer 10

1) End Replication Problem

2) dsDNA breaks happen
- chromosome ends must be distinguishable from sporadic DNA breaks
- Extensive exposed dsDNA breaks lead to cell death by apoptosis
-X-Ray Irradiation causes double stranded breaks.

Question 11

What are the characteristics of Telomeres at the end of our DNA?

Answer 11

• Non-coding sequence
• 10-15 kbp in humans
• 3’ end overhang of ssDNA
• 150-200 bases
• Hexametric repeats of (TTAGGG)
• allows us to learn a lot about cancer, how we age, etc.

Question 12

The Telomerase Holoenzyme Complex (RNA-dependent DNA Polymerase)

Answer 12

• Has an RNA and DNA component (both essential)
• TERC (abundant)
• TERT (limiting)
• In different organisms, telomerase varies in size, composition, and sequence, but certain features are conserved: all telomerase complexes contain TERC (the RNA component) and TERT (the reverse transcriptase), with TERC providing a template for telomere repeats (e.g., TTAGGG in humans, though with slight sequence variations in other organisms like yeast). In humans, telomerase also includes accessory proteins that help extend and maintain telomere ends.
• binds to DNA, positions it properly so that the end can be extended, added with the reverse transcriptase.

Question 13

Telomerase Reaction Cycle

Answer 13

• cycle is very short.
• at the core, has a primer (from the RNA component).
• the domain is the primer anchoring site, the primer is the single stranded end of our DNA, it binds and positions in the active site, so that the RT activity can extend it by 5 or 6 nucleotides. 1 repeat of telomere is added before it stops and cinches up, then it releases and can bind again and repeat the cycle, eventually it just falls off.

Question 14

Telomere Loop (T-Loop)

Answer 14

The Telomere Loop (T-loop) is a structure formed at the end of telomeres to protect chromosome ends. The single-stranded 3’ end of the telomere invades the double-stranded region, creating a closed loop. This invasion displaces a segment of DNA, forming a D-loop (displacement loop), which stabilizes the T-loop. The G-rich strand (G-strand) and C-rich strand (C-strand) in this region are also covered by protective proteins, helping to shield telomeres from degradation and improper repair.

Question 15

How could you determine Telomere length?

Answer 15

• By the Terminal Restriction Fragment Protocol (Assay)
  -Isolate genomic DNA from a cell, digest with restriction enzyme, cut most DNA around it but not the telomeres. This is over population doubling, so you don’t have telomerase activity. Start to see this smearing going downward and can see the size of the telomeres at any given time.

Question 16

How could you measure Telomerase activity?

Answer 16

• Telomere Repeat Amplification Protocol (TRAP) Assay.
• Take nuclear extract, add oligonucleotide called TS. This corresponds to telomeric repeat and binds to primer, and telomerase will add to the repeats. Let the reaction happen, allow to be extended. Add reverse primer, to assess how many repeats have been added (done by PCR), this is SCX reverse primer, and then you run on gel to see if there is activity, if there is a ladder that increases, then you see there is telomerase activity.

Question 17

What do the G-strands form?

Answer 17

• Quadraplexes
• At ends, G-rich, thought to be important to protect the ends of the telomers, and forms the G-strand quadruplexes. Very stable hydrogen bonding that forms these complexes.
  -There are antibodies that will detect these quadraplex structures.

Question 18

The Shelterin Complex

Answer 18

• It is a protein complex that binds to the ends of telomeres to protect them from being recognized as DNA damage.
• It maintains telomere integrity and stability, preventing chromosomal fusion and degradation.
• made up of 6 different proteins that interact with telomeres:
  Core proteins:
• TRF1 and TRF2 (form dimers that bind telomeric repeats, nucleate the binding of other Shelterin components)
• Tin2: acts as a bridge between TRF1 and TRF2, facilitating their interaction.
  -RAP1: binds to TRF2, playing a role in telomere protecting and length regulation.
  -POT1 (protector of telomeres): binds to a single-stranded regions of telomeres, within the D-loop.

* if does not function properly, telomeres can be misidentified as sites of DNA damage, leading to inappropriate DNA repair responses*

Question 19

Why do Shelterin complex proteins exhibit features like transcription factors? give a few examples.

Answer 19

• due to their ancient DNA-binding domains

1) Myb domain: needed for their DNA-binding capability.

2) POT1: contains an OB (oligonucleotide/oligosaccharide-binding) domain, which is conserved across many species.

3) CDC13: yeast protein like POT1, featuring two OB domains and functioning similarly to protect telomeric DNA.

Question 20

Summarize the Roles of the Shelterin Complex

Answer 20

• Protects telomeres from degradation and recognition as DNA damage.
• Maintains the structure of telomeres, preventing chromosomal fusion.
• Essential for the proper functioning of telomeres during DNA replication.
• the shorter the telomerase gets, the more accessible it is, as it gets longer, it is less accessible. A bunch of different factors control accessibility.

Question 21

Telomere Regulation by POT1

Answer 21

• The formation of a T-loop at the telomere end affects its accessibility for maintenance and repair.
• When telomeres are too short, the T-loop unfolds, making the telomere end accessible.
• If the OB domain (which binds single-stranded DNA) in POT1 is deleted, POT1 can still attach to the Shelterin complex but cannot bind the D-loop. This lack of stabilization leads to excessive telomere lengthening in the mutant. Conversely, with a stabilized D-loop, the 3’ end remains inaccessible, preventing uncontrolled telomere extension.

Question 22

Telomere Repeat Non-Coding RNA (TERRA)

Answer 22

Long Telomeres:
TERRA levels are low.
TERRA is degraded by an enzyme called RNAseH2, which helps control its levels.

Short Telomeres:
TERRA levels are higher.
TERRA forms structures called R-loops (RNA-DNA hybrids), which cause replication stress. This stress is a signal for the cell to repair and lengthen the telomeres.
The cell uses a process called Homology-Directed Repair (HDR), which helps fix the damaged telomeres and extends their length.
- associated with telomeres, crucial for telomere maintenance and regulation.

Question 23

What is an extra telomeric function?

Answer 23

is that it can bind to intergenic regions and regulate nearby gene expression.

Question 24

How are telomeres related to cancer?

Answer 24

• In humans, Telomerase is expressed at very high levels during embryonic development, but then down-regulated a few weeks after birth in most tissues.
• In humans, normal differentiated cells do not expressed Telomerase (hTERT), only cells with replicative capacity express telomerase including stem cells, skin keratinocytes, germ cells and some immune cells.
• Lack of Telomerase expression in most somatic cells is an important tumour supressor mechanism.
• Telomerase is an extremely potent oncogene (immortality enzyme - all by increasing telomere length)

Question 25

Why do cells that do not have telomerase stop growing?

Answer 25

• Hayflick Limit – he first observed this in the 50s.
• Reach senescence phase, the curve flattens out. Lead Hayflick to propose that human cells have built in mechanism where they have maximal number of cell divisions where they just stop dividing.
• Human cells stop growing due to replicative senescence.

** tumour cells bypass rpelicative senescence, Cancer cells have infinite division potential within them, there is no limitation. Very different than most cells in body that have Hayflick Limit **

Question 26

What happens when DNA damage response (DDR) triggers replicative senescence?

Answer 26

• Most cells do not have telomerase activity, which means that they will experience the end replication problem as their DNA is replicated, it gets shorter during every round of replication.
• As cells divide, see gradual decrease of telomere length through the end replication problem. Eventually reach certain length where they stop dividing (hit wall). Telomere gets short enough, the shelter complex is not there enough to protect the telomeres, and it is recognized for DNA damage response, triggering this response.

Question 27

Dysfunctional Telomeres, Senescence, and Apoptosis via Tumor Suppressor Pathways

Answer 27

1) Telomere Shortening Triggers Damage Response:

• Shortened telomeres activate damage-sensing proteins, ATM (recognizes double-stranded breaks) and ATR (recognizes single-stranded breaks).
• ATM and ATR activate CHK1/CHK2 kinases and p53, which triggers the tumor-suppressor pathway.
  p53 turns on p21, a transcription factor that mediates senescence.

2) Rb Pathway and Cell Cycle Control:

• Rb is activated by telomere shortening and regulates cell cycle arrest at the G1/S boundary.
• Rb binds E2F, a transcription factor family that promotes S-phase entry by activating genes for DNA replication (e.g., DNA polymerase, nucleotide synthesis enzymes, telomerase).
• When Rb binds E2F, it blocks S-phase entry, preventing cell division.
  Cancer Implications:
• p53 and Rb are frequently mutated in cancers.
• Mutations in upstream regulators (e.g., Ras) can inactivate Rb, disrupting its control over the cell cycle.

Question 28

p53: The Universal Tumor Suppressor

Answer 28

1) Function: p53 is a transcription factor and tumor suppressor protein, often referred to as a “universal cancer constant” because it is mutated in about half of all cancers.

2) Structure: p53 forms a tetramer (four copies) to effectively bind DNA.

3) Mutations: Most mutations are missense point mutations rather than complete deletions, making these mutations more oncogenic than simply losing p53.

4) Importance: Mice lacking p53 develop cancer rapidly, highlighting its critical role in tumor suppression.

5) Comparative Biology: Whales and elephants, which have many more cells than humans, possess multiple copies of p53, preventing widespread mutations and reducing cancer incidence.

6) Activation Triggers: p53 is activated by DNA breaks, telomere shortening (sheltin complex loss), UV light, cellular stress, and oncogenes, all of which promote senescence and help prevent cancer.

Question 29

Genomic Instability and the Bridge-Fusion-Break (BFB) Cycle

Answer 29

1) Overview: Loss of telomeres results in genomic instability through the Bridge-Fusion-Break (BFB) cycle, where the ends of chromosomes resemble DNA damage due to double-strand breaks.

2) DNA Repair Mechanism: When telomeres shorten, the cell’s DNA repair mechanisms mistakenly try to fix the broken ends. This can lead to improper joining of sister chromosomes or fusion of different chromosomes, which is detrimental when they enter the cell cycle.

3) Centrosome Tug-of-War: When two chromosomes with centrosomes are present, they engage in a tug-of-war during cell division, causing random breaks in the genome. This leads to repeated cycles of breaking and fusion, perpetuating the BFB cycle until resolution or cell death occurs.

4) Consequences: BFB cycles contribute to aneuploidy and genomic instability, which are common in cancer cells. This instability is associated with:

5) Loss of tumor suppressor genes.
Amplified growth pathways that accelerate cancer progression.
Mutations: The most common mutation in this context is in the p53 gene. However, mutations in non-coding regions, particularly those affecting telomerase, occur more frequently than p53 mutations.

6) Implications: The end products of the BFB cycle can be particularly dangerous, leading to uncontrolled cell growth and cancer development.

Question 30

How do cancer cells maintain telomerase?

Answer 30

1) Activation of Telomerase Expression

2) Alternative Lengthening of Telomere (ALT) pathway.
-> uses homologous recombination; template is donated from long telomere to short telomere to lengthen it.
-> TERRA is important for this whole process of the Alt pathway. TERRA is a scaffold and recruits the homologous recombinant machinery to the short telomere (first seen in cancer cells)
- may also drive telomere maintenance in the immune system. Antigen presenting cells donate their telomeres to the T cells. Telomeric DNA is being transferred into the T cell, so it becomes longer in the T cell.

Question 31

What are strategies for therapeutic inhibition of telomerase?

Answer 31

1) Telomerase Inhibitors
- make active site inhibitors to block the enzyme
- main drug: GRN36

2) TERT immunotherapy (getting immune system to target cancer cells)
- take antigen presenting cells from a patient and programming them with peptides or mRNAs that code antigens to try and make the immune system mount an immune response to cells that present telomerase activity.
- TERT peptides, mRNA and DNA vectors.

3) Telomere-disrupting agents
- There are drugs that can stabilize these G form quadraplexes, when that happens telomerase does not extend, and the cell undergoes senescence. These are not in the clinic yet. These seem to work quite well in vitro.

4) Block Telomerase expression or biogenesis:
- Dyskerin, part of telomerase holoenzyme complex - therapeutically inhibit telomerase.

Question 32

Telomerase & Aging

Answer 32

• A knockout mouse has been made that completely lacks telomerase activity.
• Phenotype of the Mouse… nothing!
• Until after 6 generations of mating when the mice develop aging associated disorders:
  o Male and Female Infertility
  o Alopecia and Hair Graying
  o Heart failure
  o Immunosenescence.
  o Decreased regeneration of the digestive system, skin, and hematopoietic system.
  o Early death.
  o Less prone to cancer.
• Cross section of Duodenum showing atrophy of villi.

Question 33

Human Genetic Disorders Caused by Telomerase Mutation

Answer 33

• Common one is: Dyskeratosis congenita. (not a complete loss of telomerase activity, just a partial loss)

o Skin pigmentation.
o Nail dystrophy.
o Mucosal Leukoplakia.
o Bone Marrow Failure.
o Pulmonary Disease.
o Premature loss of teeth.
o Premature hair loss/graying.
o Early death.
o Increased cancer.

• Mutations in coding sequence for telomerase reverse transcriptase
• Mutations in accessory proteins of telomerase

o Dyskerin
o NOP10
o NHP2
- Mutations will lead to disease.
o DKC
o Bone marrow failure syndromes
o Pulmonary fibrosis

Question 34

What is the mechanism of telomeric retrotransposons in drosophilia?

Answer 34

In Drosophila, telomeric retrotransposons, specifically HeT-A and TART, contribute to telomere maintenance by serving as a source of telomeric repeats. HeT-A is transcribed into RNA, which is then reverse transcribed into DNA and integrated into the chromosome ends, adding telomeric repeats that compensate for the DNA loss during replication. TART, on the other hand, can amplify HeT-A sequences, further reinforcing the telomeres. This retrotranspositional mechanism helps protect chromosome ends from degradation and prevents telomere shortening, maintaining chromosomal stability during cell division.

Question 35

What is the function of the T-loop in the telomere structure?

Answer 35

The T-loop, or telomere loop, protects chromosome ends by preventing degradation and masking them from being recognized as DNA breaks. It also regulates telomere length by influencing telomerase activity and prevents unwanted chromosome fusions, thus maintaining genomic stability.

Question 36

To the right, a terminal restriction fragment protocol was performed, Which of the following groups has the longest telomeres?

Answer 36

• length of the band/fragment indicates the length of the telomere so it would be the longest band.
• The intensity or darkness of the bands reflects the amount of DNA present.
• generally, the longest telomere will be associated with the youngest age.

Question 37

Which two proteins are activated through dysfunctional telomeres?

Answer 37

Dysfunctional telomeres activate two key proteins: ATM (Ataxia Telangiectasia Mutated) and ATR (ATM and Rad3-related).

1) ATM: This protein primarily responds to double-stranded DNA breaks and recruits the DNA damage repair machinery to address the damage caused by telomere dysfunction.

2) ATR: This protein is more specific to single-stranded DNA breaks and also helps in signaling the DNA damage response, but it functions similarly to ATM in activating downstream repair pathways.

Question 38

Explain the bridge-fusion-break cycle and how this may affect a karyotype.

Answer 38

The bridge-fusion-break (BFB) cycle occurs when dysfunctional telomeres cause chromosomes to fuse together during cell division, creating a chromosomal bridge. This tension can lead to breaks, resulting in random chromosomal fragments that may fuse again, perpetuating instability. This cycle contributes to aneuploidy and structural abnormalities in the karyotype, increasing the risk of cancerous transformations.