Telomere Notes and Other Important TTR

What are telomeres?

Telomeres are protective caps at the end of chromosomes that become shorter with each cell division.

What sequence represents telomeres?

TTAGGG

What kind of sequence is the telomere sequence?

Non coding repetitive sequence

What is the t loop?

The loop that forms with the help of the shelterin complex to keep the ends of chromosomes from sticking together and to avoid triggering DNA repair

What is shelterin?

The complex that shapes repetitive sequences into the t loop, gathers the ends of chromosomes so they cannot be mistaken for damaged DNA

6 proteins of shelterin

1. TRF1

2. TRF2

3. POT1

4. TIN2

5. TPP1

6. RAP1

Which proteins in the shelterin complex bind directly to DNA?

TRF1, TRF2, POT1

What protein is considered the bridge holding the shelterin complex together?

TIN2

Telomeric repeat binding factor 1 (TRF1)

Prevents telomerase from over extending the strand by binding to the double stranded portion of the telomere

Telomeric repeat binding factor 2 (TRF2)

Forces DNA to bend into the t loop structure by hiding the 3’ overhang

Protection of telomeres 1 (POT1)

Binds to single stranded 3’ overhang, preventing DNA damage enzyme (ATR) from seeing the exposed end and triggering senescence

TRF1 Interacting nuclear protein 2 (TIN2)

Holds everything together, the glue. Attaches TRF1 and TRF2 and recruits POT1

(TPP1)

POT1s cofactor, helps recruit telomerase if the cell needs to extend telomeres

Repressor/Activator Protein 1 (RAP1)

Binds to TRF2, to prevent homology directed repair (HDR)

What part of the chromosome does POT1 bind to?

Single stranded DNA, the 3’ overhang

What does TPP1 bind to?

TIN2 and POT1

What does RAP1 bind to?

TRF2

What does TIN2 bind to?

• TRF1

• TRF2

• TPP1

What does TRF1 and TRF2 bind to?

Double stranded DNA

• TRF2 also binds to RAP1

Why is it important that the shelterin complex forms the t-loop?

Forming the t-loop stops ATM and ATR kinases from recognizing double and single strand DNA breaks and beginning repair, to stop NHEJ which would fuse nearby chromosomes to each other

Once telomeres reach a certain length, they become too short and the cell dies during the next division. Why?  

If telomeres get too short the t-loop cannot form.

What are the functions of telomeres?  

• To prevent chromosomes ends from sticking to each other

• To prevent chromosomes from being mislabeled as broken DNA

• To provide some space for the end replication problem

Senescence

When the cell enters growth arrest, state G0 forever

Apoptosis

Cell death if the cell continues to divide anyway even though it has short telomeres

In what phase of the cell cycle are telomeres becoming shorter? 

During S phase (synthesis) telomeres are becoming shorter after replication (due to inability to replicate the very end of the lagging strand).

At what stage does the cell realize telomeres are too short? What happens next?

The cell will realize telomeres are too short at the G1 phase and either throw it into senescence or clear it for the next stage. If it clears it for the next stage, that can lead to disorders and other complications

How does telomerase work?

Telomerase uses an RNA template to synthesize and add the telomeric repeat back onto the ends of the chromosomes.  

What's the average length of human telomeres at birth?  

8,000 and 11, 000 base pairs 8-11 kb

What's the average length of human telomeres at age 35?  

(5,000 – 7,000 bp 5 – 7kb)

What's the average length of human telomeres at 65+?

(3,000 – 5,000 bp 3 – 5kb)

What's the average length of human telomeres at senescence?

(< 3,000 – 4,000 bp)

What are the factors influencing telomere length?

• Heritability – long telomeres can run in families and, some studies have shown telomere length is influenced by the fathers age.  

• Rapid early attrition – the rate of telomere shortening is most rapid during the first few years of life due to the large amount of cell proliferation required for infant growth and expansion of the immune system.  

Why do telomeres attach to the nuclear envelope?

To ensure proper chromosome positioning and keep the telomeres away from the middle of the nucleus were there is active DNA repair happening

Why use leukocyte telomere length as the standard?

Blood telomere length is the standard because it is easy to collect samples, easily accessible and is a good representative of the entire biological age of the organism

What’s typically analyzed in blood regarding telomeres and potential TBDs?

• Peripheral blood mononuclear cells  

• Granulocytes  

• Hematopoietic stem cells  

T/F: At birth telomere length is highly synchronized across different tissues, later the lengths vary more between cell types because of different rates of cell division depending on what needs to be replaced  

True

What tech is being used to measure telomeres?

Flow – FISH, southern blot or qPCR are used but Flow-FISH is the go to

Why do ATM and ATR think regular telomeres look like DNA breaks? 

Normal chromosomes end in blunt ends which to ATM looks identical to a chromosome thats been snapped in half by radiation or other types of stress. It looks like major damage.  

ATM

Ataxia – telangiectasia mutated

ATR

ATM and Rad3 related

Why is POT1 so key to stopping ATR?

• ATRs will try and “fix” the single stranded 3’ overhang always left at the end of telomeres if the overhang is not coated by POT1.

• POT1 will prevent replication protein A from binding and coating the exposed ssDNA and signaling ATR.

How can problems with blocking ATM and ATR can lead to dyskeratosis congenita

A mutation in TIN2 or TPP1 can cause the t loop to unfold and all ATM and ATR will recognize the blunt ends of the chromosome, leading to repair, resulting in apoptosis or senescence

Whats the most common type of bone marrow failure associated with dyskeratosis congenita?

Aplastic anemia

Classic diagnosis triad for DC?

1. Abnormal skin pigmentation

2. Nail dystrophy

3. Oral leukoplakia

How many estimated patients in the u.s. have the disease, what is the ratio?

1:1,000,000

~345 patients