Week 3: DNA Damage Response

Question 1

What causes DNA double stranded breaks

Answer 1

1. Caused by ionizing radiation, radio mimic chemicals, mechanical stress during replication, topoisomerase inhibitors

Question 2

What happens when you fail to repair DSBs

Answer 2

1. Failure to repair of DSB can lead to acentric and dicentric chromosomal fragments and thus genomic instability

Question 3

Protein kinase activation loop/T loop & P loop

Answer 3

1. T loop: near the substrate binding domain: kinases are activated by phosphorylation of specific residues in the T loop (oppose the positive charge of the arginine in the catalytic loop)
2. P loop: binds the substrate residue just C-terminal of the phosphorylated one (the P+1 residue)

Question 4

PIKK: DNA-PK structure

Answer 4

1. From the N-terminus, helices are arranged as HEAT repeats and folded into a hollow circular structure (concave shape)
2. After this domain, the polypeptide chain then crosses the circular structure and forms the larger C-terminal head/crown domain that is perched on top of the circular structure
   2a. This region is also predominantly α-helical and is likely to interact with many other proteins
   2b. Contained in this region is the protein kinase domain, involved in both autophosphorylation and the phosphorylation of other proteins.

Question 5

PIKK: DNA-PKcs + NHEJ

Answer 5

1. DSB induction
2. Ku loading (senses DSB)
3. DNA-PKcs recruitment, autophosphorylates
4. Recruitment of downstream NHEJ core factors
5. DNA-end ligation via DNA ligase IV
6. Ubiquitination of DNA-PKcs
7. Artemis resolves some errors
8. DNA is fixed (but error prone)

Question 6

PIKK: VDJ recombination mechanism (NHEJ + DNA-PKcs)

Answer 6

1. induction of DSBs by RAG1 and RAG2 between coding element and the recombination signal sequence
2. These DNA hairpins are recognized, processed, and repaired by NHEJ
3. Ku heterodimer forms a ring around the DNA end that can migrate into the DNA after initial binding
4. Ku bound to a DNA can attract DNA-PKCS, which gets activated
5. DNA-PKCS autophosphorylation induces a conformational change in the DNA-bound complex of Ku and DNA-PKCS.
6. After this conformational change, Artemis opens the DNA hairpins.
7. non-templated (N) nucleotides can be inserted by terminal deoxynucleotidyl transferase (TdT) or deleted via exonuclease activity
8. If the ends are compatible, they can be ligated by ligase IV
9. Causes diff combos of VDJ due to all of these factors

Question 7

VDJ recombination and mutations in NHEJ

Answer 7

1. rejoining of V(D)J recombination intermediates is impaired when mutations in NHEJ occur=severe combined immune-deficiency (SCID)
2. Example: radiosensitive SCID patients has defects in Artemis

Question 8

PIKK: ATM activation

Answer 8

1. MRN binds to DSB
2. pre-existing ATM molecules bind to MRN= auto-P =results in dimer/ oligomer dissociation & release of kinase active monomers
3. ATM Phosphorylates several substrates including H2AX (docking site)
4. Mdc1 binds to H2AX which recruits more MRN that recruits more ATM which is a cycle that amplifies the signal of ATM
5. Also p-chk2 / p53 etc.

Question 9

PIKK: How constant are ATM protein levels

Answer 9

1. ATM is a constitutively expressed protein/remains constant within cell cycle, which is held in an inactive homodimeric or higher order multimeric state
2. IATM does not undergo any change in its total protein levels or subcellular localization after DNA damage, with the only change being dimer to monomer transition and activation of its kinase activity.

Question 10

Histone modifications via ubiquitin in DNA damage (regulation of ATM response)

Answer 10

1. DNA damage=K63-linked polyubiquitination of H2A and H2AX=DNA repair response
   1a. RNF8 and RNF168 catalyze formation of K63-linked polyubiquitination chain in histones H2A and H2AX at the site of DNA damage
   1b. Polyubiquitination of H2A and H2AX facilitates the accumulation of DNA repair proteins including 53BP1 and BRCA1 at the DNA damage foci. (Wo/ this; cells sensitive to IR)
   1c. The ubiquitin modification can be removed by ubiquitin specific peptidases known as DUBs

Question 11

Homologous repair in ATM

Answer 11

1. DSBs recruit MRN complex which recruits ATM
2. Resection of DNA strand =recruitment of downstream substrates =formation of D-loop structure
3. DNA polymerases extend the resected DNA strands using homologous sequences as template which results in the formation of Holliday junction.
   3a. The Holliday junction may be resolved either in crossingover or non-crossingover fashion and the nicks are sealed by DNA ligases.

Question 12

Ataxia Telangiectasia (A-T)/Louis-Barr syndrome characteristics

Answer 12

1. A-T is a recessive disorder caused by mutations in ATM gene
2. The disease is characterised by ataxia (uncoordinated body movements and telangiectasia, which means enlarged blood capillaries that can be seen under the skin), immune deficiency, hypersensitivity to ionizing radiation, and a predisposition to certain cancers, sterility in aldulthood, above normal intelligence
   2a. About 20 percent of those with A-T develop cancer, most frequently acute lymphocytic leukemia or lymphoma.
3. Patients show severe radiosensitivity whereby normal doses of chemotherapy, to cure cancer caused by AT disease, to be lethal.

Question 13

Why do people w A-T have disorders

Answer 13

1. When DNA damage, neural stem cells (NS) proliferate but neural progenitors (NP) and lineage-restricted precursors (RP) should undergo programmed cell death via ATM signalling to p53 to activate apoptosis. In A-T, due to an absence of ATM or p53, the cells differentiate along all lineages and survive. Therefore, in the setting of catastrophic DNA damage, loss of ATM, results in enhanced survival of neural progenitors.

Question 14

PIKK: ATR mechanism

Answer 14

1. SSB recruits RPA which recruits ATRIP which recruits ATR
2. ATR phosphorylates Chk1 and RPA
   2a. Chk1 phosphorylation: fast
   2b. RPA: is progressively phosphorylated by ATR during SSB resection
3. ATR is activated at collapsed replication forks and it’s goal is to stop replication
4. Nbs1 is important for this pathway to function!

Question 15

Inhibition of cell cycle via inhibiting CDK1/2

Answer 15

1. Via ATR/ATM
   1a. ATR and ATM are sensors of SSBs and DSBs, respectively, which activate CHK1 and CHK2, respectively, by phosphorylation.
   1a. CHK1 and CHK2 =inhibition of CDK1 and CDK2 (preventing the removal of inhibitory phosphates placed on CDK1 and CDK2, by WEE1)
2. DNA damage=p53 activated (via phosphorylation by ATM and CHK2) =inhibit cell-cycle progression by promoting transcription of p21, which in turn inhibits the kinase activity of the cyclinE–CDK2 complex.