Genomic instability and DNA repair

Question 1

What kind of screen can be used to test for synthetic lethality in a gene-mut cell line?

Answer 1

Use a library of siRNAs to suppress gene product transcription, and test to see if cells die. If they do, suppression of library gene and original mut gene are synthetically lethal

Question 2

Hallmarks of cancer

Answer 2

1. Sustaining proliferative signaling: too much in healthy cells -> senescence
2. Evading growth suppressors
3. Evading apoptosis
4. Limitless Replicative Potential
5. Sustained Angiogenesis
6. Metastasis
7. Reprogrammed energy metabolism
8. Evading Immune Destruction

approximately 6 specific enabling mutations must occur to make out of a normal cell a tumor cell

Question 3

Enabling characteristic

Answer 3

1. Genetic and epigenetic instability

2. Inflammation

Question 4

Mutations

Answer 4

Small: missense/nonsense substitution, frame-shit
large: translocation, deletion, insertion, amplification

Mutations in oncogenes are generally dominant, whereas mutations in tumor suppressor genes are recessive.

Question 5

Monoclonicity

Answer 5

The vast majority of human tumors is monoclonal. But: genetic heterogeneity which is due to genetic instability may mask the true monoclonal origin.

Originally shown in leukemia with IgG specificities.

Question 6

p53

Answer 6

• “master guardian”
• senses DNA damage, cell cycle abnormalities, hypoxia.
• normally repressed by mdm2.
• is a transcription factor
• can either halt cell cycle progression (DNA repair or senescence) or induce apoptosis.
• binds as tetramer.
• Vast majority of mutation are missense (dominant-negative hypothesis), affecting DNA-binding region.

Activated by:

• DNA damage signals,
• Oncogene signaling,
• lack of nucleotides (too much anabolism)
• hypoxia (too much metabolism, poorly matured blood vessels)

Question 7

20/20 rule

Answer 7

• Oncogenes: >20%of recorded mutations are at recurrent positions and are missense.
• Tumor suppressors: >20%ofthe recorded mutations in the gene are inactivating.

Question 8

Measuring p53 DNA-binding

Answer 8

In vitro: radio labeled binding, visualization with gel chromatography.
In vivo: Chip-seq. crosslinking, sonication, Ab for DNA-associated proteins, sequencing.

Question 9

Regulation of p53

Answer 9

Each monomer bound by MDM2 to inhibit. MDM2 is transcription is activated by p53 (negative feedback). MDM2 binding -> p53 ubiquitination.

Question 10

Downstream of p53

Answer 10

• > Bax (pro-apoptotic factor on mitochondria)

- > p21 –| cycE,A, Cdk2 -> RB, E2F -> cc arrest

Question 11

DNA photodamage

Answer 11

• UV rays cause pyramidine dimers

- Ionizing radiation causes ROS which cause DNA/protein linkages or d/ssDNA breaks, and base damage

Question 12

DNA repair mechanisms

Answer 12

Excision:

• BER: Base excision repair
• MMR: Mismatch repair
• NER: Nucleotide excision repair
• Ribonucleotide excision repair

Low fidelity DNA polymerases-Translesion polymerases

Double strand break repair

• NHEJ: Non homologous end-joining
• MMEJ: Microhomology directed end-joining (or Alt-EJ)
• HR: Homologous recombination (BRCA 1/2)

Question 13

Endogenous ds Break formation

Answer 13

Topoisomerases, hydrolysis: AP sites, oxidative metabolism -> ssDNA breaks. 1% of these are left unrepaired and become dsDNA breaks during replication.

Question 14

dsDNA repair strategies

Answer 14

• NHEJ leads to deleted DNA sequence at break, mutagenic, cycle independent.
  1. protein capping of ends.
  2. ligatable ends: DNA-PKcs, Artemis cleave overhangs
  3. Polλ/μ synthesis
• HR requires DNA template, G2/S phase.

-MHEJ: limited asymmetric preprocessing of DNA (Dna2, MRN), annealing & synthesis (Polθ).
MMEJ is an error-prone method of DNA repair and results in deletion mutations

Question 15

Homologous recombination procedure

Answer 15

1. DNA resection (RPA coated, then BRCA2 exchanges RPA for Rad51)
2. d-loop invasion of template (Rad51 helps with strand invasion).
3. polymerization on both strands, double holliday junction formation.
4. vertical or horizontal cleavage may lead to reciprocal crossover (-> sister chromatid exchange)

Question 16

PARP1

Answer 16

• BRCA-deficient cells rely heavily on PARP1. .
• Binds to ssDNA breaks, promotes base-excision repair and micro- homology mediated end joining
• PARPi leads to ssDNA breaks becoming dsDNA breaks.
• Can also trap PARP on DNA, very toxic, prevents repl. fork movement.

Question 17

ht-RNAi screens:

Answer 17

Can detect synthetic lethality, gene products leading to drug resistance and thus targets that would increase drug effectiveness.

Question 18

Why are PIR (parp-inhibitor resistant) clones also resistant to cisplatin, but not to docetaxel?

Answer 18

PIR clones restore HR function by restoring the reading frame of a truncated protein. Cisplatin causes DNA adducts and stalled replication forks, which can be repaired using HR. cisplatin is therefore dependent on an incompetent HR pathway, whereas docetaxel is cytotoxic through a completely independent pathway (microtubule stabilizing).

Question 19

Mechanisms of PARPi resistance

Answer 19

1. Restoration of HR, ex. through 2º mutation.
2. loss of PARP1 expression.
3. PARPi pumped out of cells.

Question 20

What genes enhance effectiveness of PARPi inhibition when silenced?

Answer 20

Genes related to nucleotide excision repair

Question 21

Xeroderma pigmentosum

Answer 21

• due to deficiencies in NER
• 1000x risk to develop skin cancer
• Complementation group, must be -/- in one group to lead to phenotype.

Question 22

NER procedure

Answer 22

1. Distortion recognition (helical distortion)
2. Formation of open structure
3. Dual incision at 3’ and 5’ end of open structure and damage location
4. Excision of 24-32 bp.
5. DNA repair synthesis & ligation

recruitment of NER proteins can be visualized though fluorescent protein-tagged protein and spot UV treatment.

Question 23

Transcription Coupled Repair

Answer 23

Actively transcribed strand of gene is preferentially repaired. TCR lost in Cockayne’s syndrome. Premature aging (12 y life span, decrease in txn after UV damage)

Question 24

Base Excision Repair

Answer 24

• Glycosylases remove aberrant bases
• AP endonuclease, phosphodiesterase remove sugar & phosphate backbone.
• DNA polymerase adds new nucleotide, ligase seals nick.
• also repairs abasic sites.
• Lack of pathway leads to embryonic lethality, cancer.

Question 25

Why do rad9-mut yeast not die immediately after irradiation w/ UV, but continue to replicate and then die?

Answer 25

Rad9 is a DNA damage sensor, so the cell does not stop to repair damage, and accumulates it instead.

Question 26

Checkpoint activation by DNA lesions

Answer 26

ds break: ATM activation, relocalization
phosphorylation (dimer -> active monomer). activates p53, Creb, BRCA1, Chk2

ss break: ATR relocalization -> Chk1
ATR-ATRIP localizes to ssDNA break where it phosphorylates substrates.

ATR is a drug target in cancer. ATRi -> mitotic catastrophe.

Question 27

Chk1/2

Answer 27

Checkpoint kinases, activated by ATM, ATR. Activate p53, and inhibit mdm2, leading to cell cycle arrest or apoptosis.

Question 28

S Phase checkpoints

Answer 28

Replication checkpoint, S-M checkpoint, Intra-M checkpoint. Inhibit ‘Late’ origin of replication or mitosis.

Question 29

Ataxia Telangiectasia

Answer 29

Sensitivity to ionizing radiation
Spontaneous chromosome breaks
Radioresistant DNA synthesis

–>
Severe immonodeficiency
Progressive Ataxia due to degeneration of cerebellum
(ataxia: lack of coordination of muscles)
Telangiectasia
Lymphomas
Carcinomas

Question 30

Seckel Syndrome

Answer 30

ATR: ATM- and RAD3- related Hypomorphic alleles

--> 
Microcephaly (small circumference of the head) Dwarfism
Large eyes, low ears, small chin
Severe mental retardation
chromosome breaks

Question 31

γH2AX

Answer 31

• colocalized with BRCA1 at ds DNA breaks.
• modified histone protein. A direct consequence of DNA damage, and needing to access DNA template around break, more loosely associated to DNA

Question 32

Methods for identifying protein-protein interactions

Answer 32

• coimmunoprecipitation, problem: Can coprecipitate proteins that are not in the same compartment
• Y2H: Prey and Bait bind. Bait linked to DNA-binding protein for upstream motif. Prey linked to activator for reporter gene.
• BioID method: Interacting proteins covalently labeled with biotin. Allows harsh processing, ID of weak/transient interaction, ID of environment.

Question 33

Detecting native protein size

Answer 33

• size exclusion chromatography: smaller molecules enter bead pores, take longer to elute.
• sucrose gradient ultracentrifugation.

Question 34

Hela biology

Answer 34

Does not necessarily reflect normal cell biology, especially when discussing checkpoints. True of any immortalized line.

Question 35

Ataxia Telengiesctasia

Answer 35

Related to loss of S phase DNA checkpoints. Main consequence: DNA damage during synthesis phase doesn’t stall replication, leading to a large amount of DNA errors. Radioresistant DNA synthesis.