Genomic instability and DNA repair Flashcards
(35 cards)
What kind of screen can be used to test for synthetic lethality in a gene-mut cell line?
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
Hallmarks of cancer
- Sustaining proliferative signaling: too much in healthy cells -> senescence
- Evading growth suppressors
- Evading apoptosis
- Limitless Replicative Potential
- Sustained Angiogenesis
- Metastasis
- Reprogrammed energy metabolism
- Evading Immune Destruction
approximately 6 specific enabling mutations must occur to make out of a normal cell a tumor cell
Enabling characteristic
- Genetic and epigenetic instability
2. Inflammation
Mutations
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.
Monoclonicity
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.
p53
- “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)
20/20 rule
- Oncogenes: >20%of recorded mutations are at recurrent positions and are missense.
- Tumor suppressors: >20%ofthe recorded mutations in the gene are inactivating.
Measuring p53 DNA-binding
In vitro: radio labeled binding, visualization with gel chromatography.
In vivo: Chip-seq. crosslinking, sonication, Ab for DNA-associated proteins, sequencing.
Regulation of p53
Each monomer bound by MDM2 to inhibit. MDM2 is transcription is activated by p53 (negative feedback). MDM2 binding -> p53 ubiquitination.
Downstream of p53
- > Bax (pro-apoptotic factor on mitochondria)
- > p21 –| cycE,A, Cdk2 -> RB, E2F -> cc arrest
DNA photodamage
- UV rays cause pyramidine dimers
- Ionizing radiation causes ROS which cause DNA/protein linkages or d/ssDNA breaks, and base damage
DNA repair mechanisms
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)
Endogenous ds Break formation
Topoisomerases, hydrolysis: AP sites, oxidative metabolism -> ssDNA breaks. 1% of these are left unrepaired and become dsDNA breaks during replication.
dsDNA repair strategies
- 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
Homologous recombination procedure
- DNA resection (RPA coated, then BRCA2 exchanges RPA for Rad51)
- d-loop invasion of template (Rad51 helps with strand invasion).
- polymerization on both strands, double holliday junction formation.
- vertical or horizontal cleavage may lead to reciprocal crossover (-> sister chromatid exchange)
PARP1
- 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.
ht-RNAi screens:
Can detect synthetic lethality, gene products leading to drug resistance and thus targets that would increase drug effectiveness.
Why are PIR (parp-inhibitor resistant) clones also resistant to cisplatin, but not to docetaxel?
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).
Mechanisms of PARPi resistance
- Restoration of HR, ex. through 2º mutation.
- loss of PARP1 expression.
- PARPi pumped out of cells.
What genes enhance effectiveness of PARPi inhibition when silenced?
Genes related to nucleotide excision repair
Xeroderma pigmentosum
- due to deficiencies in NER
- 1000x risk to develop skin cancer
- Complementation group, must be -/- in one group to lead to phenotype.
NER procedure
- Distortion recognition (helical distortion)
- Formation of open structure
- Dual incision at 3’ and 5’ end of open structure and damage location
- Excision of 24-32 bp.
- DNA repair synthesis & ligation
recruitment of NER proteins can be visualized though fluorescent protein-tagged protein and spot UV treatment.
Transcription Coupled Repair
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)
Base Excision Repair
- 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.
