Telomerase/DNA Damage

Question 1

What is the end replication problem?

Answer 1

Okazaki fragment on lagging strand will cause a 3’ overhang once RNA primer is released. This will cause sequential loss of chromosomal end every time replication occurs.

Question 2

How to overcome end replication problem:

Answer 2

circular chromosome, end recombination, telomere transposition, genome concatamerization, hairpin ends, terminal protein primer, telomere repeat addition

Question 3

How do you look at telomeres?

Answer 3

FISH (Fluorescence in situ hybridization)

Question 4

What binds at telomere ends?

Answer 4

Shelterin complex

Question 5

TR Function

Answer 5

Telomerase RNA, provides 1 & 1/2 repeat as template

Question 6

What is TERT?

Answer 6

Telomerase Reverse Transcriptase

Question 7

How is TR conserved across species?

Answer 7

no primary sequence conservation, only SOME secondary structures are conserved
i.e. Core, CR4/CR5 stem

Question 8

What happens if p53 is activated?

Answer 8

Leads to apoptosis/senescence

Question 9

What is H2AX?

Answer 9

histone variant that is involved in DNA damage signaling

Question 10

Histone Chaperones

Answer 10

Change subunits of nucleosomes throughout the genome

Question 11

MRN Function

Answer 11

Bind ds DNA breaks and recruit ATM

Question 12

ATM function

Answer 12

recruited by MRN and phosphorylates H2AX

recruited for DNA double strand breaks

Question 13

MDC1 Function

Answer 13

binds to phosphorylated H2AX and recruits 53BP1

Question 14

53BP1 Function

Answer 14

Recruited by MDC1, activate Chk2

Question 15

ATR Function

Answer 15

Analogous to ATM, recruited by replication stress, UV; activated Chk1

Question 16

CHK1/2 Function

Answer 16

P-CHK1 - triggered downstream of ATR
P-CHK2 - triggered downstream of ATM

phosphorylate CDC25A to inactive protein/phosphorylate p53 to allow to to escape MDM2

Question 17

CDC25A Function

Answer 17

Dephosphorylate CDK1 and Cyclin, allows G1-S transition

Question 18

MDM2 Function

Answer 18

bind p53, inactivates it

Question 19

p53 Function

Answer 19

phosphorylated version causes transciptional activation of p21

Question 20

CDK1-Cyclin B Function

Answer 20

enables G1-S transition

Question 21

DNA Break H2AX Phosphorylation Pathway

Answer 21

1) MRN binds double stranded break
2) MRN recruits ATM
3) ATM phosphorylates H2AX
4) Phosphorylated H2AX binds MDC1
5) MDC1 recruits 53BP1

Question 22

p21 Function

Answer 22

Inactivates CDK1-cyclin B

Question 23

G1-S Cell Cycle Regulation through CDC25A

Answer 23

1) ATM (ATR analogous) gets activated.
2) Downstream effect -> CHK1/2 get phosphorylated and activate
3) P-CHK1/2 inactivate CDC25A by phosphorylation
4) P-CDC25A cannot dephosphorylate CDK1-Cyclin B
5) CDK1-Cyclin B is inactive, cannot trigger G-S transition

Question 24

G1-S Cell Cycle Regulation through p21

Answer 24

1) ATM (ATR analogous) gets activated
2) Downstream effect is CHK1/2 get phosphorylated and activated
3) p53 gets phosphorylated, disassociates from MDM2, which inactivates p53
4) p53 transcriptionally activates p21 gene
5) p21 inhibits CDK1-cyclin B

Question 25

What type of telomere dysfunction will activate DNA damage response?

Answer 25

Loss of telomere binding protein, altered telomere repeat sequence, telomere shortening

Question 26

What is ALT?

Answer 26

Alternative Lengthening of Telomeres

Occurs in Telomerase-null yeast
Strand invasion followed by polymerase filling in

Question 27

How do you measure telomere length?

Answer 27

q-FISH

Question 28

How many generations of mice does it take to see affects of telomerase KO?

Answer 28

4-5 generations

Question 29

What is genetic anticipation?

Answer 29

Earlier onset and severity of disease in sequential generations. Occurs in families with mutant telomerase.

Question 30

How did researchers test if telomerase activity is important in cancer cells?

Answer 30

Use tumor prone mouse and cross with mice that have short telomeres. Look at mice across generations and around at around G6, see mice living a lot longer

Question 31

How are telomerase levels increased in cells?

Answer 31

Myc amplification, loss of MenI (repressor), TERT/TR amplification, TERT rearrangement, TERT promoter mutations

Question 32

What happens if telomeres are too short or continuously maintained?

Answer 32

Too short: Stem Cell Failure

Too long: Cancer