Nepveu Lectures (Repair Lectures 1-4) Flashcards
What are the four major threats to the integrity of DNA?
1) Lesions inflicted by endogenous metabolites.
-> Reactive Oxygen Species (ROS).
-> Spontaneous deamination (cytosine, adenine, guanine, and 5’ methyl cytosine)
2) Lesions caused by environmental DNA-damaging events.
-> UV.
-> y-radiations.
-> Chemical mutagens.
3) Replications errors which have escaped the editing process during DNA synthesis.
4) Errors made during DNA repair.
What are responses to DNA damage that are specific to some organisms?
There is conservation of DNA repair mechanisms through evolution.
In microorganisms, there are inducible repair functions.
-> SOS system (counteracts the effects of DNA-damaging agents, this reflects their existence in hostile environmental niches)
-> Adaptive mutagenesis.
Bacteria can also elevate their mutation frequency to adapt to a new environment. This inducibility is absent in mammalian cells, one exception is lymphoid B cells, where somatic hypermuation within immunoglobin genes contributes to antibody maturation.
In higher organisms, there is apoptosis and cellular senescence.
What are the different DNA Repair Pathways?
1) Direct reversal of damage: MGMT, accepts methyl groups from O6-methyl guanine.
2) Base Excision Repair (BER): most DNA lesions from endogenous sources.
-> endogenous DNA lesions arising and repaired in a diploid mammalian cell = intrinsic mutational process as a result of normal metabolism: depurination, oxidation, alkylation of bases are frequent. Then you would do BER to repair this.
3) Nucleotide Excision Repair (NER): bulky lesion causing structural distortions.
4) Mismatch Repair: errors made during replication.
5) Double-strand break repair pathways.
-> Non-homologous end joining (NHEJ).
-> Homology dependent repair (HR).
6) Damage tolerance.
-> Template switching (post-replication repair)
-> Lesion bypass (translesion synthesis)
7) Cell cycle checkpoint control
8) Apoptosis
9) Senescence
What are major sites of cellular reactive oxygen species (ROS)? (Cellular Redox Homeostasis)
ROS (Fenton reaction) do not travel because they are reactive, but can be converted into peroxide, which travels, once in contact with ferrous ions, can be converted to hydroxyl radicals that can cause damage to various molecules, including DNA.
1) Mitochondrial electron transport chain (Mito-ETC).
2) Endoplasmic retculum (ER) system.
3) NADPH oxidase (NOX) complex.
Damaging agent: UV light or polycylic aromatic hydrocarbons.
What is DNA lesion and the repair pathway associated?
DNA Lesion:
- 6-4 photoproduct
- Cyclobutane pyrimidine dimer
- Bulky adduct
Repair pathway:
- Nucleotide excision repair (NER)
Damaging agent: X-rays, Oxygen Radicals, Alkylating agents, spontaneous.
What is DNA lesion and the repair pathway associated?
DNA Lesion:
- Abasic site; oxidized, deaminated, alkylated bases.
- DNA ss break.
Repair pathway:
- Base excision repair (BER).
Damaging agent: Ionizing radiation, Hydroxy urea, UV light, X-rays, Anti-cancer agents.
What is DNA lesion and the repair pathway associated?
DNA Lesion:
DNA ds break.
(can also cause DNA ss break and abasic base site)
Repair pathway:
DS break repair
-> HR
-> NHEJ
Damaging agent: Replication & Recombination errors
What is DNA lesion and the repair pathway associated?
DNA Lesion:
Base mismatches
Insertion
Deletion
Repair pathway:
Mismatch repair (MMR).
There are a few diseases caused by inherited mutations that impair the BER. What would the result of these be?
Embryonic Lethal.
What is the mechanism of BER (Base Excision Repair)? (what are the key players?)
1) BER initiated by removal of modified base by either a monofunctional or bifunctional DNA glycosylase to leave an abasic site (AP).
Pathway A)
Excision:
-> Monofunctional (UDG) (MPG)
Makes AP.
Incision:
5’ APE1 to DNA backbone.
End processing:
PolB or APE1 or PNKP. (depending on specific nature of terminus)
Pathway B)
Excision:
-> Bifunctional (NHT1)(OGG1) (NEIL1)(NEIL2)
Makes AP.
Incision:
3’ AP lyase B-elimination.
NTH1/OGG1 -> APE1 (end-processing)
NEIL1/NEIL2 -> PNPK (end-processing)
Now both pathways have same steps:
Then goes through either short-patch BER/SSBR or long-patch BER/SSBR.
Short patch:
- repair synthesis of the single-nucleotide gap by Pol-B with XRCC1 then DNA ligation by LIG3 and then repair is done.
Long patch:
- repair synthesis of 2-13 nucleotide gap by Pol-B and/or Pol delta or epsilon aided by PINA and RFC.
-resulting 5’ flap removed by FEN1.
-final ligation by LIG1.
In BER, what happens if single-strand breaks occur by other means?
They can contain simultaneous 3’ and 5’ obstructive termini.
-> PARP1 recognizes these breaks and start performing ADP-ribosylation, & end processing takes place, uses TDP1 and APTX.
What is the connection between base excision repair and transcription?
- Some TFs function as accessory factors in BER.
- They stimulate enzymatic activities of BER enzymes.
- DNA de-methylation is performed by BER.
- MPG interacts with RNA pol II through direct interaction with the elongation complex. Active transcription elongation promotes efficient MPG-directed repair.
- Some BER enzymes can recruit a TF close to a promoter.
What are the two pathways of mammalian nucleotide excision repair?
1) GC-NER: global genome excision repair.
2) TC-NER: transcription-coupled nucleotide excision repair.
- In TC-NER, Single Strand Break protein travels with Pol II and recruits DNA repair proteins when RNA Pol II stalls at site of DNA damage.
What is the mechanism of Nucleotide Excision Repair (NER)? What are the key factors?
(1) DNA Damage Recognition
- Helix distortion damage takes place.
- There are two pathways, GC-NER and TC-NER.
Pathway (1) GC-NER:
- recognized by XPC complex ((DDB1, DDB2) known as XPE), facilitate recognition of lesions.
- XPC complex recruits TFIIH to the repair site.
Pathway (2) TC-NER:
- recognized by the stalling at RNA at Pol II, facilitated by CSB, CSA, XAB2.
- CSA recruits TFIIH to the repair site.
(2) DNA Unwinding.
- XPD and XPB subunits of TFIIH are DNA helicases that unwind the DNA in the immediate vaccinity of the lesion.
- RPA and XPA bind to keep the DNA strands apart.
(3) For dual incision:
- XPA recruits XPF-ERCC1 endonuclease to incise the damaged DNA strand 5’ to the lesion while XPG incises 3’ to it.
(4) Repair synthesis:
- performed by DNA polymerase delta and kappa or epsilon with help of accessory proteins RFC, PCNA, RPA.
(5) DNA Ligation:
- LIG1 or LIG3a-XRCC1
In terms of TC-NER, what is a feature of CS that has to do with this pathway?
*** normal human cells preferentially repair the transcribed strand of active RNA polymerase II transcribed genes. Cells from patients affected by Cockayne Syndrome (CS) do not show this preferential repair. Two complementation groups have been identified for CS: CS-A and CS-B.
What are human diseases caused by defects in NER?
1) Xeroderma Pigmentosum (XP)
- characterized by clinical photosensitivity
- XP-A and XP-G complementation groups identified.
2) Cockayne’s syndrome (CS)
- sun-sensitive, distinctive array of congenital neurological and skeletal abnormalities.
- CSA & CSB.
3) Trichothiodystrophy (TTD)
- share many symptoms with CS, definitive symptoms is brittle hair & nails.
- mutations in at least 3 TFIIH genes are implicated in TTD: XPD, XPB, TTDN1
** fibroblasts derived from patients with these UV sensitive syndromes have been instrumental in identifying proteins involved in NER pathway.
What is the mechanism of Mismatch Repair in E.coli? What initiates this mechanism?
It is initiated by mistakes during replication due to slippage of DNA replication machinery along the DNA template in regions of simple repeat sequences.
(1) MutS
(2) MutL & MutH bind + ATP
This leads to the introduction of distant strand break in the newly synthesized DNA strand.
(3) MutH recognizes hemimethylated d(GATC) sequence and cleaves the strand.
(4) UvrD protein, DNA helicase, unwinds the DNA from the nick, the displaced DNA strand is degraded by exonuclease + ssb (protecting remaining strand).
(5) gap filling by DNA Pol II & joining by DNA ligase completes the repair process.
What is the mechanism of mismatch repair (MMR) in humans?
(1)
- Recognition & binding of mismatch by MSH2-MSH6 or MSH2-MSH3.
- Recruits MLH1-PMS2 complex (form ternary complex).
(2)
- Excision by 5’ to 3’ exonuclease EXO1. RPA binds to protect SS-DNA during excision.
(3)
- repair synthesis is accurately performed by Pol Delta.
(4) Ligation of the remaining nicks after synthesis is by LIG1.
What is implicated in determining the direction of mismatch repair in mammalian cells?
- PCNA.
In Human MMR, what happens if you need to do 3’ directed MMR?
- endonuclease function of PMS2 is activated by ‘ nick, stimulated by RFC & ATP, to make the 5’ nick, the excision can follow by Exo1.
What is Gene Conversion? What is co-conversion?
- Gene conversion happens during DNA repair when two similar but slightly different DNA sequences exchange genetic information.
- If there is a mismatch (base pairing error) between the two DNA strands in a region called heteroduplex, the cell’s MMR system comes to fix it.
- Since each heteroduplex region has 2 strands, there are two heteroduplex regions per recombination event, there are four possible ways mismatches can be repaired.
- Co-conversion: if there are two mismatched sites in the heteroduplex DNA that are close together, the mismatch repair system might fix both sites during same repair event.
- Gene conversion can happen with or without recombination. With, the sequence on either side of the heteroduplex swap places between the two DNA moleciles. Without, only heteroduplex regions is affected, regions on either side stay the same.
Homeologous Recombination
- Recombination between two DNA molecules that diverge at many positions is inhibited by MMR pathway.
- The capacity to perform homeologous recombination is believed to help pathogenic bacteria to acquire new traits.
How is a single oxidative DNA lesion repaired vs a clustered oxidative DNA lesion? What causes them?
- They are caused by Ionizing Radiation.
- Single is repaired by BER.
- Clustered is repaired by BER, NHEJ, and NER.
These are all DNA repair mechanisms.
What are 3 ways that double-stranded breaks are produced? (how are each repaired?)
(1)
DBS’s generated when the two complementary strands of DNA are broken simultaneously at sites that are sufficiently close to one another, that base-paring and chromatin structure are insufficient to keep two DNA ends juxtaposed.
Repaired by HDR & NHEJ.
(2)
Replication through a region with SSB can cause a DSB (“double-strand end”-DSE)
Repaired by HDR.
(3)
Repair of damaged bases that are close to one another can generate “Secondary DSBs.”
What is the model for double-stranded break repair?
(A)
Recombination repair pathway (homology-dependent repair) (HR)
- involves nucleolytic resection of one strand followed by strand invasion of a homologous DNA molecule (sister chromatic and DNA repair synthesis).
- sister chromatic is used as a template to replace the genetic information lost during the nucleolytic process.
(B)
Non-homologous end-joining pathway (NHEJ)
- Kv heterodimer binds to DNA ends and recruits DNA-PKcs.
- may be repaired accurately or inaccurately.
- goes through re-ligation or fill-in/deletion ligation.
What original observations led to DSB repair model?
- key idea is that a DSB in DNA is a trigger for homologous recombination.
1) Circular plasmid.
2) Linearized plasmid.
3) Linearized plasmid with a deletion.
What is the mechanism of Double-stranded break repair (DBS repair) for HR (homology-dependent repair)?
(1)
Recognition of double-stranded break.
- MRN complex recruited to break, binds to DNA through MRE11.
- RAD50, ATM, NBS1, BRCA1, 4H2AX, MDL1.
- RNF8 -> initiated ubiquitylation cascade of histones H2A & H2AX causing chromatin restruction and generation of binding sites for further protein factors.
(2)
End-processing:
- damaged DNA ends are processed to prepare for repair.
- CtIP starts process by performing 5’ resection.
- this relies on CtIps recruitment of BRCA1.
- more resection carried out by EXO1, producing SS-DNA with 3’ overhangs.
(3) Nucleoprotein filament formation:
- RAD51 monomers bind to ss-DNA 3’ overhangs, forming RAD51 nucleoprotein filaments.
(4)
RAD51 recombinase complex assembly.
- built with accessory proteins (BRCA 1/2, RAD52, RAD51 paralogs)
(5)
Homology search, strand invasion & D-loop formation.
- Rad51 recombinase complex & RAD54 search for homologous DNA sequence in sister chromatid/homologous chromosome.
- strand invasion occurs, forming D-loop.
(6)
DNA repair synthesis.
- DNA polymerase uses homologous strand in D-loop as template to extend 3’ invading strand through DNA repair synthesis.
-> branch migration -> holiday junction.
-> capture of second DNA end -> double holiday junction (DHJ).
- LIG1 seals
- structure-specific endonuclease cut junctions to produce either:
- cross-over products (swapped DNA regions).
- non-cross over products.
- the BLM RecQ helicase with TOPO3a & BLAP65, can make non-crossover products.
What is the mechanism of Double-stranded break repair (DBS repair) for NHEJ (non-homologous end joining)?
(1)
Recognition of double-stranded break.
- MRN complex recruited to break, binds to DNA through MRE11.
- RAD50, ATM, NBS1, BRCA1, 4H2AX, MDL1.
- RNF8 -> initiated ubiquitylation cascade of histones H2A & H2AX causing chromatin restruction and generation of binding sites for further protein factors.
(2)
End binding.
- Ku70-Ku80 heterodimer binds to broken DNA ends. (protects DNA termini)
(3)
DNA-PK complex formation.
- DNA-PKCS recruited to DNA ends by Ku70-Ku80.
- Forms the DNA-PK complex, holds two broken DNA ends close together.
(4)
End processing and resection.
- DNA-PKCS autophosphorylates.
- Processing enzymes TDP1, FEN1, WRN helicase, artemis.
- Produce 5’-P & 3’-OH termini required for ligation.
(5)
DNA-repair synthesis.
- DNA polymerase fill in any missing nucleotides.
- DNA-PK then recruits LIG4-XRCC4-XCF complex.
- LIG4 seals DNA ends (ligation).
Why did cells develop Damage Tolerance mechanisms?
Because lesions that persist hamper the replication apparatus, cells have evolved a damage tolerance system to allow complete replication in the presence of DNA damage. This response tolerates, rather than removes, DNA damage, and consists of at least two mechanisms.
What are the two mechanisms cells have developed for ‘Damage Tolerance?”
1) Template switching.
2) Lesion Bypass (translesion synthesis).
What are factors that contribute to the persistence of DNA damage?
a) high levels of damage.
b) poorly repaired lesions.
c) inefficiently repaired genomic regions.
d) DNA damage occurred during the S phase of the cell cycle.
What is Template Switching?
“post-replication repair”
- avoids replication of the damaged site of the DNA template.
- by using the newly synthesized daughter strand as template (template switching), the replicative machinery effectively circumvents the lesion block DNA and proceeds with replication.
What is Lesion Bypass? (translesion synthesis)
- directly utilizes the damaged template.
- can be divided into two steps:
i) nucleotide incorporation opposite the lesion (i.e., translesion synthesis), followed by
ii) extension of DNA synthesis.
After a short stretch of extension, normal DNA synthesis by the replication apparatus can then resume.
Error-prone lesion bypass constitutes the major mechanism of DNA damage-induced mutagenesis in cells.
What does lesion bypass require?
- specialized DNA polymerases (Pol) that can use damaged DNA as template: the Y family (UmuC superfamily): Umu for “umutable.”
*** lesion bypass can either be error-free or error-prone.
What does translesion synthesis ensure?
Completion of DNA replication.
Domain Structure of Y-family DNA polymerases
- the gene names Umu (unmutable) in E.coli and Rev (reversion-negative) in yeast refer to the fact that mutants in these genes exhibit a reduced rate of mutations.
- Indeed, most mutations in bacteria and yeast are caused by the action of translesion synthesis polymerase.
What are the key factors at play for:
The SOS response to UV induction in E.Coli?
1) Binding of LexA repressor to regulatory operators upstream of SOS genes (limits their expression under normal growth conditions)
2) RecA induced from UV, activated by binding to regions of ssDNA to form nucleoprotein filament, RecA*
3) RecA* coprotease in autocleavage of LexA → induction of SOS genes.
4) Proteolytic processing event, UmuD, forms UmuD’, two form with one UmuC to form polV (2 UmuD’ + 1 C).
What is Adaptive Mutagenesis? (biochemical properties of error-prone polymerases)
- Refers to a process whereby mis-adaption of bacteria to a new environmental conditions (lack of nutrients, change in temperature, antibiotics) triggers the induction of error-prone polymerases whose action increases mutation frequency).
- This process produces a highly heterogenous population of genetic variants, increasing the probability of obtaining a clone that can proliferate in new conditions.
- This process does not exist in higher organisms, although an analogous process takes place during somatic hypermutation in B cells.
(somatic mutation is one example where specialized cells purposely increase the frequency of mutations in a specific genomic locus)
TLS Polymerases in Somatic Hypermutation
1) Purpose of SHM: SHM generates mutations in immunoglobulin genes to create antibodies with higher affinity for antigens.
2) Initiation by AID:
Activation-induced cytidine deaminase (AID, yellow oval) initiates SHM.
AID deaminates cytosine (C) in DNA, converting it to uracil (U) (red).
3) Role of Transcription:
RNA polymerase II (RNA Pol II, brown oval) exposes single-stranded DNA during transcription, providing access for AID.
4) Mutations at C-G Pairs:
–> Replication over uracil:
Replicating over uracil causes C → T or G → A mutations (transition mutations).
–> Error-prone repair by Ung:
Uracil DNA glycosylase (Ung, blue oval) removes the uracil, leaving an abasic site (F).
Error-prone repair by translesion synthesis polymerases (Rev1, yellow square, or TLS pol, purple diamond) can cause transition or transversion mutations (labeled as ‘N’).
5) Mutations at A-T Pairs:
–> Mismatch recognition by Msh2/Msh6:
The U-G mismatch is recognized by mismatch repair proteins Msh2/Msh6 (green and purple ovals).
These recruit exonuclease 1 (Exo1, yellow triangle) and polymerase eta (Polh, pink oval), spreading mutations to nearby A-T base pairs (also labeled as ‘N’).
6) High-Fidelity Repair:
–> BER and MMR pathways:
Ung and Msh2/Msh6 can function in normal base excision repair (BER) or mismatch repair (MMR), accurately repairing the uracil without introducing mutations.
What Ensures Fidelity during RNA Replication?
- In a WT-strain, the serial action of base selectivity, proofreading and mismatch repair reduces the error rate of DNA replication to a low level.
- In a mismatch-repair-defective phenotype (mutL), the error rates represent the efficiency of base selection and proofreading.
- In a double-mutant for mismatch repair and proofreading (mutDmutL), the error rates represent the efficiency of base selectivity only.
In adaptive mutagenesis (stress-induced mutagenesis) in micro-organisms, what is a contributing factor to the higher rate of mutations?
- the up-regulation of PollV and PolV.
- the inhibition of mismatch repair.
If there is a single-stranded break or base damage, what repair mechanism is used and what proteins are used in that repair mechanism?
You would use BER.
DNA glycosylases.
APE1.
PolB.
PARP1.
XRCC1.
LIG3
or
long patch repair:
DNA pol epislon/delta
LIG1.
If there is a double stranded break, what repair mechanism is used and what proteins are used in that repair mechanism?
HDR:
- MRN complex
- Ctlp
- RAD51 complex (RAD51/BRCA1/BRCA2/RAD52)
NHEJ:
-MRN complex
- Ku70
-KU80
-DNA-PK
-WRN-RecQ
If there is Guanine methylation, what repair mechanism is used and what proteins are used in that repair mechanism?
MGMT
If there is a pyrimidine dimer or bulky adduct, what repair mechanism is used and what proteins are used in that repair mechanism?
NER
1) GG-NER:
XPC
XPE
XPB
XPD
RPA
XPA
XPF-ERCC1
XPG
DNA Pol epsilon/delta/k
LIG1
2) TC-NER:
CSA
CSB
If there is a base mismatch/insertion/deletion, what repair mechanism is used and what proteins are used in that repair mechanism?
Mismatch repair
E.coli:
MutS
MutL
MutH
Mammals:
MSH2/MSH6
MSH2/MSH3
MLH1-PMS2
Exo1
DNA Pol epsilon
LIG1
When there is a DNA damage response, what happens?
Senses the DNA damage and initiates kinases, which then will initiate effector kinases.
Initiating Kinases:
ATM
ATR
DNA-PK
Effector Kinases
CHK1 (by ATM)
CHK2 (by ATR)
Ataxia telangiectasia mutated (ATM)
Ataxia-telangiectasia (AT) is a human autosomal recessive genetic disorder which is characterized by symptoms including immune deficiencies, neuronal degeneration, growth retardation, and an approximate 100-fold increase in the incidence of cancers.
Ataxia telangiectasia and Rad 3 related (ATR)
Like ATM, ATR is a nuclear protein kinase. The protein kinase activity of the ATR/Rad3 (MEC1) homologues is essential for mediating checkpoint regulation.
DNA-dependent protein kinase (DNA-PK)
Mice that carry inactivating mutations in either Ku70, Ku80 or DNA-PK are defective in DSB repair. These mutants are hypersensitive to ionizing radiation, defective in V(D)J recombination and suffer from severe combined immunodeficiency (SCID).
What does DNA damage involve?
- multiple types of post-translational modifications and complex signalling cascades.
What is the difference between Ubiquitination and SUMOylation at PCNA at the Replication fork?
(1)
Sumolyation of PCNA prevents recombination during replication.
(2)
Mono-ubiquitination leads to translesion synthesis, and poly-ubiquitination promotes template switching.
G1/S and G2M Checkpoint Controls
Both checkpoints involve the activation of the ATM and ATR kinases, which phosphorylate and activate the checkpoint kinases, CHK1 and CHK2.
These kinases phosphorylate and inactivate the G1/S and G2/M phosphotases, Cdc25A and Cdc25C.
In the absence of these phosphotases, cyclin E/CDK2 and cyclin B/CDK1 remain phosphorylated and inactive.
Another target of the ATM/ATR kinases in G1 is the p53 tumour suppressor. Phosphorylated p53 can activate transcription of the gene coding for CDK inhibitor, p21WAF/CIP1.
What takes place at the S Phase Checkpoint Control?
*checkpoint-induced degradation of the CDC25A phosphatase.
After a DNA double-strand break (DSB) generation and ATM activation, the rate of phosphate incorporation into CDC25A increases through combined action of CHK1 and CHK2.
This leads to stronger interaction with ubiquitin ligase, reduction of CDC25A half life, and S-phase delay.
The Mitotic Checkpoint
Spindle Assembly Checkpoint (SAC): Prometaphase
(1)
- During prophase/early prometaphase, kinetochore assembly recruits the spindle assembly checkpoint (SAC) complex to unattached kinetochores.
- SAC generates a diffusible “wait anaphase” signal that inhibits/sequesters CDC20, an activator of the anaphase-promoting complex/cyclosome (APC/C).
- Separase, the protease that cleaves cohesins holding sister chromatids, is inhibited by securin binding.
The Mitotic Checkpoint
Spindle Assembly Checkpoint (SAC):
Anaphase
(3) Following silencing of the signaling at each kinetochore and turnover of the inhibitor that transmits the wait anaphase signal, APC/C-mediated ubiquitylation (Ub) of securin and cyclin B1 and subsequent degradation by the proteosome triggers anaphase entry.
The Mitotic Checkpoint
Spindle Assembly Checkpoint (SAC):
Metaphase
(2) Methaphase
As each pair of sister kinetochores attaches to kinetochore microtubules (MT, green), and microtubule motors generate tension that stretches them, generation of the checkpoint inhibitor is silenced at those kinetochores.
Selective Pressures Imposed on Cancer Cells
1) Tumour maintenance and tumour progression require that cancer cells surmount many obstacles and adapt to selective pressures of several types.
2) A single tumour cell with a static genome cannot successfully survive these multiplication steps.
3) “Success” in tumour maintenance and tumour progression necessitates that a tumour cell population will be large enough and evolves rapidly enough to be able to generate variants that will survive the next selection step.
The Mutator Phenotype
The concept that cancer cells exhibit a mutator phenotype (mutate at a high rate) states that the large number of mutations in tumour cells cannot be accounted for by the low mutation rates of normal somatic cells, but instead must be a manifestation of a mutator phenotype.
“Cancer cells have way more mutations than normal cells. Normal cells mutate at a very low rate, so there’s no way that all those cancer mutations could happen just by chance. Instead, cancer cells seem to have something special going on that makes them mutate a lot faster. This high mutation rate is called a mutator phenotype—basically, cancer cells are “mutation machines.””
Genomic Rearrangements
- Aneuploidy
- Copy number variations (chromosome loss, amplifications, deletions)
What is the difference between point mutations oncogenes vs point mutations in tumour suppressor genes?
1) Oncogenes:
Often missense mutations.
2) Tumour suppressor genes:
More often nonsense and frameshift mutations.
What is the difference between cancer families and somatic mutations?
(1)
Cancer families:
Exhibit a predisposition to develop certain types of cancer.
Members of these families typically have inherited a germ-line mutation in a tumour suppressor gene (ex.BRCA1, BRCA2, HNPCC).
(2)
Somatic mutations:
Acquired during the person’s life.
What is Loss-of-heterozygosity (LOH)?
When one allele is deleted such that only one allele remains.
The remaining allele may or may not be inactivated by a point mutation, a short deletion or transcriptional silencing.
Knudson two-hit tumour suppressor gene hypothesis vs Haploinsufficient tumour suppresor genes.
Stipulates that the two alleles of a tumor suppressor gene must be inactivated (by deletion, point mutation or silencing) to produce a cancerous phenotype. In contrast, in the case of haploinsufficient tumor suppressor genes, inactivation of only one allele is sufficient to confer a cancerous phenotype.
In familial retinoblastoma, a person is born with one defective copy of the Rb gene in all cells. If the remaining normal copy gets mutated in a retinal cell, that cell loses Rb function and can develop into a tumor.
In sporadic retinoblastoma, both copies of the Rb gene must be mutated in the same cell for a tumor to form. Since the chance of mutating one copy is already rare, the chance of mutating both is extremely low, making it unlikely to happen in many cells. This usually results in only one tumor.
There are three categories when discussing the cellular lineage between a Fertilized Egg and a Fully Malignant Cancer Cell. What are they?
(1)
Intrinsic mutation process:
- endogenous DNA damage
- errors during DNA repair
- translesion synthesis
(2)
Environmental & lifestyle exposure:
- smoking
- UV irradiation (farmers)
(3)
Mutator phenotype
- inactivation of DNA repair
- more ROS
- Tetraploidy & chromosome
- merotely
- Chromothrypsis
- Kategis
- AID & APOBEC
(4)
Chemotherapy & radiotherapy
therapy-related leukemia.
What provided the first evidence of a Mutator Phenotype?
Microsatellite Instability (MSI) , observed in tumours which inactivating mutations in mismatch repair genes (ex. hMSH2, hMLH1).
These tumour cells not only acquire more mutations in microsatellite sequences but also in coding sequences.
Mice homozygous for KO mutations of the Msh2, MIh1, or Pms2 genes exhibit MSI and a predisposition to cancer.
What do individuals with hereditary non-polyposis colorectal cancer (HNPCC) carry?
A germline mutation in one allele of a mismatch repair gene.
Tumours are formed from cells in which the remaining normal allele is inactivated.
Kataegis
(Greek for ‘‘shower’’ or ‘‘thunderstorm’’) a phenomenon whereby point mutations rapidly occur and cluster over hundreds (microcluster) or millions (macrocluster) of DNA bases.
Chromothripsis
a phenomenon whereby individual chromosomes are shattered and reassembled in a single catastrophic event. The mechanism for chromothripsis starts with the physical isolation of chromosomes in aberrant nuclear structures called micronuclei. This is followed with the fragmentation and subsequent reassembly of a single chromatid during the next cell division.
Catastrophic chromosomal breakage -> attempted chromosomal repair -> progression towards cancer.
Some rare cells can adapt to high levels of reactive oxygen species (ROS). What are the two mechanisms to do this?
(1)
Increasing antioxidant production to balance ROS levels and protect the cell from damage.
(2)
Speeding up the repair of oxidative DNA damage, allowing cells to avoid senescence (aging) and keep dividing, even in a high-ROS environment.
Proteins like Sestrins help in this process by regenerating peroxiredoxins, which are crucial for reducing ROS and maintaining the cell’s antioxidant defenses.
What is the outcome of elevated levels of ROS in cancer cells?
- Increase the frequency of mutations.
- High ROS is another mechanism to generate a mutator phenotype.
Tetraploid Cells and Mitotic Adaptation in Cancer
- Tetraploid cells can form through:
Cytokinesis failure
Cell fusion
Mitotic slippage
Resulting in supernumerary centrosomes (extra centrosomes in the cell).
-In normal cells:
Extra centrosomes cause chaotic multipolar mitosis, often leading to non-viable daughter cells.
-In some cancer cells:
Extra centrosomes can be clustered into two poles, promoting bipolar mitosis.
These cells overexpress spindle assembly checkpoint (SAC) proteins, giving them a strong SAC that extends mitosis.
The robust SAC allows cells to successfully cluster centrosomes and divide, even with extra centrosomes.
- Non-oncogene addiction:
This adaptation is not due to reliance on a single oncogene, but rather the cancer cells’ ability to adapt to the presence of extra centrosomes through mitotic regulation, specifically by manipulating the SAC and centrosome clustering. This is an example of mitotic adaptation rather than addiction to an oncogene.
How do extra centrosomes promote genetic instability and then tumour progression?
- Multipolar spindle intermediates promote merotelic attachment.
- Metastases may often derive from primary cancer cells that are teraploid.
Therapy-Related Cancers and Their Risks
- Therapy-related cancers are new cancers that develop after successful treatment for a primary cancer.
- Cancer survivors have a significantly higher risk of developing a second primary cancer compared to the general population.
- These cancers can arise due to various treatments, such as radiotherapy and alkylating agents, and the risk is often dose-dependent.
Relative risk patterns vary for different therapies:
1) For Hodgkin lymphoma patients, the risk of developing acute myeloid leukemia (AML) peaks 2–7 years after treatment with chemotherapy.
2) Radiogenic breast cancer risk from radiotherapy peaks 15–20 years post-treatment and remains elevated thereafter.
3) Lung cancer risk after radiotherapy for Hodgkin lymphoma is dose-dependent and increases linearly with radiation dose.
Radiotherapy
- cancer treatment
- ionizing radiations will cause oxidative DNA damage, including base damage, single-strand breaks and double-strand breaks.
Chemotherapy
All the agents used cause mutations in DNA (alkylating agents, topo inhibitor) or target DNA metabolism (antimetabolite) or chromosome segregation (antimicrotubule agents).
- Antimicrotuble agents
- Alkylating agents
- Antimetabolites
- Topoisomerase Inhibitors
What is an example of resistance of cancer cells to treatments?
- For example, glioblastoma cancer cells that express higher levels of MGMT, either because of gene amplification or overexpression, exhibit resistance to the alkylating agent, temozolomide.
How was precision (personalized medicine) established?
The paradigm for “personalized medicine”, also called precision medicine, was established through research on breast cancer. Only the HER2 breast cancer subtype responds to trastuzumab.
Synthetic Lethality
Two genes are synthetic lethal only when their simultaneous inactivation results in cellular or organismal death. In this example, deletion of either gene A or gene B does not affect viability whereas inactivation of both at the same time is lethal.
What is a synthetic lethality screen?
Have cancer cells and normal cells.
1) Infect with pools of lentiviruses expression shRNAs.
2) Quantify shRNAs in each cell population.
3) Genes synthetic lethal in cancer cell.
What is the Synthetic Lethality Window?
The synthetic lethality window (or therapeutic window) refers to the range of gene inhibition where mutant cells are selectively killed without affecting wild-type cells.
What are stress phenotypes of cancer cells & non-oncogene addiction?
These include metabolic stress, proteotoxic stress, mitotic stress, oxidative stress, and DNA damage stress.
These stresses generate additional pressures that select for extensive adaptations in cellular processes that are not themselves oncogenic. Yet, cancer cells are acutely dependent on heightened expression or activity of some normal proteins. Hence, the term: non-oncogene addiction.
Non-oncogene addiction
The idea that cancer cells become unusually dependent on certain normal genes that are not themselves cancer-causing (not classical oncogenes). These genes are part of pathways that help cells cope with stress, and while healthy cells can survive without them, cancer cells rely on them to survive and grow in their stressful environment.
Therapeutic application
A practical application of this concept is that it offers additional therapeutic targets that could be exploited in cancer treatment.
Therapeutics that interfere with the functions of “non-oncogenes” could display synthetic lethality with cancer cells.
the PARP1 Paradigm (synthetic lethality)
- DNA lesions are typically repaired by multiple pathways that promote survival.
- Synthetic lethality occurs when all repair pathways are blocked, leading to cell death.
-Single-strand breaks (SSBs) are repaired by the PARP1-dependent pathway before replication forks arrive.
- If SSBs are not repaired before the fork hits, they turn into double-strand breaks (DSEs).
- DSEs at the replication fork are repaired by template switching, requiring BRCA1/2.
- BRCA1/2-defective tumors cannot repair DSEs at collapsed forks, leading to dependency on PARP1 repair.
- PARP inhibitors cause synthetic lethality in BRCA1/2-defective tumors because SSBs cannot be repaired in time, and DSEs cannot be fixed.
- Normal tissue without bi-allelic BRCA1/2 defects is not affected by PARP inhibitors.
