DNA damage & genomic instability

Question 1

What are the 2 new strands synthesized in a replication fork?

Answer 1

1. Leading strand
2. Lagging strand
   Both need RNA primer and DNA polymerase 3

Question 2

What are Okazaki fragments?

Answer 2

Parts of the newly synthesized lagging strand

Question 3

What are the 2 endogenous sources of DNA damage?

Answer 3

1. Cell replication (DNA polymerase)

2. Cell metabolism (oxidative stress)

Question 4

What are the exogenous sources of DNA damage?

Answer 4

Smoke, UV, ionizing radiation, chemicals, etc.

Question 5

How does a cell decide how to act on DNA damage?

Answer 5

It depends on the severity of the DNA damage

Question 6

What can be said on direct repair for repair of damaged bases?

Answer 6

1. Carried out by single specific enzymes
2. Sensor and effector in-one enzyme
3. Takes care of a small portion of lesions
4. Error-free

Question 7

What can be said on NER (nucleotide excision repair) for repair of damaged bases?

Answer 7

• TC-NER (transcription-coupled):

• repair of lesions that stall transcription
• mutations result in Cockayne syndrome (rapid aging)

• GG-NER (global genome):

• repair of lesions throughout the genome
• mutations result in Xeroderma pigmentosum (high UV sensitivity)

Question 8

What can be said on MMR (mismatch repair) for repair of replication mistakes?

Answer 8

‣ Removal of nucleotides (by endonuclease 1) mismatched from DNA polymerases and insertions/deletions resulting during replication or recombination
‣ Error-prone

Question 9

What can be said on BER (base excision repair) for repair of single strand DNA breaks (SSBs)?

Answer 9

‣ Repair of SSBs and single lesions such as oxidized nucleotides and apurinic/apyrimidinic sites)
‣ Short patch for the removal of single nucleotides
‣ Long patch for removal of 2 to 13 nucleotides

Question 10

What can be said on NHEJ (non-homologous end joining) and HR (homologous repair) for repair of double stranded DNA breaks (DSBs)?

Answer 10

‣ NHEJ repairs DSB fast via direct ligation independent of sequence context (error-prone), often the pathway of choice
‣ HR repairs DSB error-free, but needs undamaged DNA template and DNA end resection

Question 11

What can be said on Alt-EJ (alternative end joining) and SSA (single strand annealing) for repair of double stranded DNA breaks (DSBs)?

Answer 11

Alt-EJ or SSA are alternatives for HR, use DNA resection and DNA repeats, and thus are always mutagenic

Question 12

What is the most toxic DNA lesion?

Answer 12

DSBs (double stranded DNA breaks) are the most toxic DNA lesions and need to be repaired

Question 13

What are the 4 types of double stranded DNA breaks (DSBs) repairs?

Answer 13

1. Homologous Recombination Repair (HR) => error free
2. Non-Homologous End Joining (NHEJ) => error prone
3. Alternative End Joining (alt-EJ) => mutagenic
4. Single-strand annealing (SSA) => mutagenic

Question 14

Are double stranded DNA breaks perfect DNA repair mechanisms?

Answer 14

DSB repair can leave mutations and large deletions, specially when using alt-NHEJ (alternative end joining) or SSA (single strand annealing)

Question 15

What are the 4 tools to study DNA damage and repair?

Answer 15

1. Reporter assay: assess the efficiency of HR (homologous repair) and NHEJ (non-homologous end joining)
2. Comet assay: determine global DNA damage
3. Flow cytometry (FACS): determine cell cycle
4. Immunofluorescence/Immunoblotting (Western Blot): protein phosphorylation, recovery after laser-induced damage, DNA replication kinetics (measure replication fork progression) and recruitment of repair factors to DNA damage

Question 16

4 things to say about traditional chemotherapy VS tailored treatments to exploit

Answer 16

1. Traditional chemotherapy:
‣ target rapidly dividing cells
‣ usually cause more DNA damage 
‣ major side effects
‣ cancers often become resistant

2. Tailored treatments to exploit: 
‣ target cells with specific DDR (DNA damage response) deficiencies 
‣ usually do not cause more DNA damage 
‣ overcome resistance
‣ less side effects (hopefully)

Question 17

How can we exploit the DDR (DNA damage response) to treat cancer using replication stress (RS)?

Answer 17

Healthy cells have low RS
Tumor cells have low to moderate RS
If we inhibit cell cycle checkpoints (like ATR or Chk1) with drugs it will lead to high levels of RS and lead to cell death
(Tumor cells are much more dependent to cell cycle checkpoints than healthy cells so they will be more targeted by the drug) => I think…

Question 18

What can errors during DNA repair lead to?

Answer 18

Errors during DNA repair can cause mutations, loss of

chromosomes, DNA replication stress or cell death, posing a serious health risk to all humans (cancer and aging)

Question 19

What is DDR (DNA damage response)?

Answer 19

The DDR is a complex signaling network that ensures proper DNA repair, proliferation arrest and/or activation of cell death