Telomeres

How many telomeres and centromeres are on each chromosome?

2 telomeres

1 centromere

What are telomeres?

Physical ends/caps of eukaryotic chromosomes

What are telomeres made up of?

Specific DNA sequences and proteinss

What are the 2 main functions of telomeres?

1. Allow replication of extreme ends of the linear chromosome

2. Protect the ends of the DNA molecules = prevent enD-end fusion

How do telomeres protect the chromosome ends?

Prevent then ends being recognised as a DSB

What is the end replication problem?

The gap on the end of the lagging strand, left because the RNA primer cannot be replaced with DNA since the DNA pol needs to add to a primer sequence

What 3 things are needed for DNA replication?

Primer

Template

5'-3' direction

What are the 4 model organisms for studying telomeres?

Ciliated protozoa

Yeast

Mice

Mammalian cells in culture

What are ciliates?

Large single celled eukaryotes with elaborate cell structure

-have cilia on surface

Why are ciliates useful for studying telomeres?

Because they have so many micronuclei = all chromosomes have 2 telomeres = can purify lots of telomeric DNA

How many nuclei do ciliates have?

1 big macronucleus

1 small micronucleus

What are the chromosomes like in the micronucleus?

Typical + small

- many genes, linear, v. long

What are the chromosomes like in the macronucleus?

Unusual + big

-one gene, linear, v. short

What is amplified in the macronucleus?

Mini-chromosome

What happens when ciliates have sex?

1. Swap micronuclei

2. Undergo meiosis

3. Segregate new micronuclei

4. DNA in micronuclei is chopped up + amplified

5. Produces macronuclei

What kind of overhang is produced in a macronucleus?

3' overhang

What organism was used to define the basic features of telomeric DNA?

Tetrahymena macronuclear rDNA

Is telomeric DNA sequence organisation conserved in evolution?

Yes

What is the structure of telomeric DNA like?

Many repeats of a short unit

What is telomeric DNA rich in, running 5'-3' towards chromosome end?

TG

What is the sequence of Tetrahymena telomeric DNA?

TTGGGG

200 bp

What is the sequence of human telomeric DNA?

(TTAGGG)n

1-20kb

What is telomerase DNA rich in?

GC

In what direction does the G rich strand run in organisms with telomerase?

Towards chromosome end

Do Tetrahymena telomeres work in yeast?

Yes

What was Blackburns and Szostak's experiment with Tetrahymena and yeast telomeres?

They made a linear plasmid with Tetrahymena telomeres and inserted it into yeast

What happened to the DNA at the very end of the linear plasmid inserted into yeast?

No longer tetrahymena, but yeast telomeric DNA

What mechanism does not use pre-existing DNA template to synthesise DNA?

Telomerase

What is the template for telomerase?

Telomerase RNA

How do you perform the assay for telomerase allowed purification?

1. Start with primer from tetrahymena (TTGGGG)4

2. Add d*GTP (radioactively labelled) + dTTP

3. Produces (TTGGGG)4(TTGGGG)n

What does the assay for telomerase allowed purification produce?

Long radioactive G rich strand of telomeric DNA

How do you size fractionate the telomeric DNA produced from telomerase assay?

Use gel electrophoresis

What did Blackburn now have from the gel electrophoresis of telomerase?

Assay for telomerase

What are the 2 components of telomerase?

RNA component/template

Protein

What is the protein component of telomerase responsible for?

The catalytic steps

Why must the protein component be a RT?

Because it is using RNA as a template

What are the 5 steps in how telomerase works?

1. Telomerase binds to the telomere

2. RNA component aligns with existing telomere repeats

3. Telomerase synthesises new repeats using its own RNA as template

4. Telomerase repositions itself on the chromosome

5. RNA template hybridises with the DNA

What is the protein component of telomerase?

telomerase reverse transcriptase (TERT)

What are EST mutants in yeast?

Extra Short Telomeres

- EST 1,2,3 genes

What happens to EST mutants in yeast?

Senescent prematurely

What is the evidence that protein TERT works in vivo?

TERT protein from Euplotes

EST mutants from yeast

-EST2 lined up with TERT sequence (homologous)

-EST2 encoded the Euloptes telomerase RT

What do Reverse Transcriptases do?

Copy sequence information from RNA to DNA

What does p123 equal?

EST2 = TERT

What happens if there are mutations in the RT domain of EST?

Causes yeast to die/senesce

What is the RNA component of telomerase?

TER, TERC, TR

What does the RNA component of telomerase contain?

1.5 copies of the CnAn telomere repeat

What does the RNA component of telomerase act as?

A template for TnGn synthesis

In vitro what is required for TERT activity?

TER and TERT/EST2p

What is required for TERT activity in yeast, in vitro?

TER and TERT/EST2p & EST1p + EST3p

Name some proteins that are required for telomerase to be able to find the telomere?

NOP10, NHP2, dyskerin

How does telomerase TRT associate with telomeres?

Through specific interactions with TPP1

How does telomerase prevent shortening ends problem?

By adding telomeric unit repeats to ends

What happens to cells lacking telomerase?

Telomeres gradually shorten

What kind of primer is required by telomerase?

3' end of telomeric RNA