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

DNA damage response (DDR) overview (delete)

Answer 3

1. DSB
2. DSB recognition by specific detectors initiates the assembly of protein complexes and involve covalent modifications of proteins
   2a. The immediate early action (within minutes) of these protein complexes is to stimulate DNA repair
   2b. Cell cycle progression is halted and gene expression is altered
   2c. gene expression-dependent biological responses are delayed by a few hours: processes include inflammation, necrosis, apoptosis, differentiation, senescence, & growth arrest

Question 4

PIKK complexes (delete)

Answer 4

1. Three main complexes centred around PIKKs are responsible for detecting and coordinating the responses to double stranded breaks
2. The activity of the these kinases is responsible for initiating the cell cycle arrest, gene expression and DNA repair responses in the cell
3. These kinases are DNA-PKcs, ATM & ATR

Question 5

Protein kinase activation loop/T loop & P loop

Answer 5

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 6

PIKK: DNA-PK structure

Answer 6

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 7

PIKK: DNA-PKcs + NHEJ

Answer 7

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 8

Non-homologous end joining (delete)

Answer 8

1. DSB occurs and HR fails (no sister chromatid available)
2. NHEJ can now occur
3. Resection of single strands by exonuclease
4. DNA strands brought together using a template via base pairing of the processed end of the DNA: possible limited base pairing between them
5. Strands filled in; joined by ligation
6. Double helix reconstruction
   5a. NHEJ has errors and sequence deletions

Question 9

What is V(D)J recombination used for: (delete)

Answer 9

1. Genes that encode active immunoglobulins (Ig) and T-cell receptors (Tcr) since they are not not present in the germ line.
2. Ig and Tcr genes are assembled in B and T cells, respectively

Question 10

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

Answer 10

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 11

VDJ recombination and mutations in NHEJ

Answer 11

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 12

PIKK: ATM activation

Answer 12

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)
   2c. Mdc1 binds to H2AX which recruits more MRN that recruits more ATM which is a cycle that amplifies the signal of ATM

Question 13

PIKK: How constant are ATM protein levels

Answer 13

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 14

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

Answer 14

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 15

What happens when you knockdown histone ubiquitination enzymes (delete)

Answer 15

1. impairs DSB-associated polyubiquitination of H2A and H2AX
2. inhibits retention of 53BP1 and BRCA1 at the DSB sites,
3. sensitizes cells to ionizing radiation

Question 16

PIKK: Activation of ATM (delete)

Answer 16

1. DSB=relaxation of chromatin=involves the recruitment of the MRN complex to break
2. ATM is then recruited to the site of the break by the MRN complex and phosphorylates members of the complex and other downstream substrates.
3. Acetylation (Ac) also contributes to the process of activation.
   3a. The acetyltransferase TIP60 is constitutively associated with ATM, and in the presence of a DNA DSB it becomes activated and acetylates ATM at Lys3016 within the C-terminal FATC domain.
4. XRCC4, the requisite cofactor of DNA ligase 4 and NHEJ is detected at the break site after ATM recruitment

Question 17

Homologous repair in ATM

Answer 17

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 18

Regulation of homologous repair (delete)

Answer 18

1. DSBs=ATM=P-H2AX & P-MDC1=polyubiquitin chains on histone 2A.
2. Molecules recruit BRCA1=DNA repair response

Question 19

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

Answer 19

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 20

Why do people w A-T have disorders

Answer 20

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 21

PIKK: ATR (Delkete)

Answer 21

1. ATR is an apical kinase in one of the DNA-damage induced checkpoint pathways

Question 22

PIKK: ATR mechanism

Answer 22

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 23

Nbs1 in ATR (delete)

Answer 23

1. the role of Nbs1 in RPA phosphorylation: ne
2. An Nbs1 mutant that is unable to bind RPA fails to support proper recovery of collapsed replication forks, suggesting that the Nbs1-mediated mode of ATR activation is important for the repair of replication-associated DSBs.

Question 24

Cell cycle control

Answer 24

1. ATR, ATM, CHK1, CHK2, and WEE1 inhibit cell-cycle progression into S-phase and mitosis following DNA damage.
2. This regulation occurs ultimately through the control of the cyclin-dependent kinases (CDK) that facilitate entry into the S- and M-phases of the cell cycle, namely CDK2 and CDK1.
3. The activity of CDK1 and CDK2 are controlled by phosphorylation and their regulatory cyclin subunits.

Question 25

Inhibition of cell cycle via inhibiting CDK1/2

Answer 25

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.