Telomerase/DNA Damage Flashcards
Lets learn about telomerase and DNA damage
What is the end replication problem?
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.
How to overcome end replication problem:
circular chromosome, end recombination, telomere transposition, genome concatamerization, hairpin ends, terminal protein primer, telomere repeat addition
How do you look at telomeres?
FISH (Fluorescence in situ hybridization)
What binds at telomere ends?
Shelterin complex
TR Function
Telomerase RNA, provides 1 & 1/2 repeat as template
What is TERT?
Telomerase Reverse Transcriptase
How is TR conserved across species?
no primary sequence conservation, only SOME secondary structures are conserved
i.e. Core, CR4/CR5 stem
What happens if p53 is activated?
Leads to apoptosis/senescence
What is H2AX?
histone variant that is involved in DNA damage signaling
Histone Chaperones
Change subunits of nucleosomes throughout the genome
MRN Function
Bind ds DNA breaks and recruit ATM
ATM function
recruited by MRN and phosphorylates H2AX
recruited for DNA double strand breaks
MDC1 Function
binds to phosphorylated H2AX and recruits 53BP1
53BP1 Function
Recruited by MDC1, activate Chk2
ATR Function
Analogous to ATM, recruited by replication stress, UV; activated Chk1
CHK1/2 Function
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
CDC25A Function
Dephosphorylate CDK1 and Cyclin, allows G1-S transition
MDM2 Function
bind p53, inactivates it
p53 Function
phosphorylated version causes transciptional activation of p21
CDK1-Cyclin B Function
enables G1-S transition
DNA Break H2AX Phosphorylation Pathway
1) MRN binds double stranded break
2) MRN recruits ATM
3) ATM phosphorylates H2AX
4) Phosphorylated H2AX binds MDC1
5) MDC1 recruits 53BP1
p21 Function
Inactivates CDK1-cyclin B
G1-S Cell Cycle Regulation through CDC25A
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
G1-S Cell Cycle Regulation through p21
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
What type of telomere dysfunction will activate DNA damage response?
Loss of telomere binding protein, altered telomere repeat sequence, telomere shortening
What is ALT?
Alternative Lengthening of Telomeres
Occurs in Telomerase-null yeast
Strand invasion followed by polymerase filling in
How do you measure telomere length?
q-FISH
How many generations of mice does it take to see affects of telomerase KO?
4-5 generations
What is genetic anticipation?
Earlier onset and severity of disease in sequential generations. Occurs in families with mutant telomerase.
How did researchers test if telomerase activity is important in cancer cells?
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
How are telomerase levels increased in cells?
Myc amplification, loss of MenI (repressor), TERT/TR amplification, TERT rearrangement, TERT promoter mutations
What happens if telomeres are too short or continuously maintained?
Too short: Stem Cell Failure
Too long: Cancer
