Genomic Instability Flashcards
What does cancer generation require?
Mutation
What is genomic instability?
The increased acquisition of genomic alterations
What leads to genomic instability?
Defects in mechanisms to protect genomes
What are the two general types of DNA damage?
Exogenous and Endogenous
List some examples of exogenous damage
UV, x-rays, chemical
List some examples of endogenous damage
Metabolism, replication errors, fork stalling
How can the byproducts of metabolism result in oxidative DNA damage?
Byproducts include reactive oxygen species with unpaired electron (eg peroxide, hydroxyl radical, superoxide anion) can cause altered bases and strand breaks etc as they can alter chemical composition of DNA
State the 2 ways DNA is protected
It’s structure and detoxification
How is DNA protected by its structure?
Housed in nucleus so is protected from physical/ chemical damage
Tightly packed, wrapped around his tones, folded into fibres > coils > chromosomes which reduces damage
How is DNA protected by detoxification?
Harmful agents are neutralised. Eg:
- via redox pathway
- cell membrane pumps which pump our harmful agents
- enzymes which catalyse harmful agents into safe products
- proteins which conjugate harmful chemicals before they come into contact with cell
How does the redox pathway help protect DNA?
Superoxide radical (O2-) is converted to H2O2 by the action of a super-oxide dismutase. This can be converted to a damaging hydroxyl radical which damages DNA, but is instead converted to H2O and O2 by catalase or into 2H2O via glutathione peroxide, both of which are not harmful to DNA
List the classes/types of DNA damage
- loss of base
- small additions to nucleotides (adducts)
- bulkier adducts
- single or double strand breaks
- mismatched bases
- cross links in DNA within and between strands
Describe the most common example of small adduct DNA damage
Most common in 8oxo-G where oxygen is added to the 8th carbon of guanine under oxidative stress. Changes structure of guanine
What happens if small adducts go unrepaired?
Base pairs in corrects, eg 8-oxoG to A instead of C
Causes mismatch during replication
Describe bulky adducts and give an example
Big changes in structure
Eg: benzopyrene (polycyclic aromatic hydrocarbon) present in polluted air, food, cigarette smoke, is a big adduct onto guanine
What happens if a bulky adduct goes unrepaired?
Can distort DNA helix which blocks replication and stalls transcription
Describe single strand breaks
Loss of a single nucleotide, 5’ and 3’ termini damaged at end of breaks
What happens if a single strand DNA break goes unrepaired?
Can block DNA replication and stall transcription
What is the difference between double strand and single strand breaks?
Double strand loses bases in both strands so there is so template to copy lost info from unlike single strand breaks.
What happens if double stranded breaks go unrepaired?
Risk loss of genetic information during replication
Describe cross-link DNA damage
Covalent link formed between nucleotides. Can be:
- intrastrand: between neighboring nucleotides
- interstrand: between opposite nucleotides
What happens if cross-link DNA damage goes unrepaired?
Can cause stalling of replication forks resulting in loss in genetic material
Why is important to protect DNA in non-dividing and dividing cells?
Nondividing: damages accumulate
Dividing: block DNA replication, fixed as mutation which can be passed onto daughter cells, may result in cancer
What are the 3 things that may happen to a cell with accumulated DNA damage?
Becomes senescent, apoptotic, or cancerous
What happens if an error is detected by the complex signalling networks?
Cell cycle arrest initiated
DNA repaired by DNA pathway repairs
Transcriptional program activation or apoptosis
At which cell cycle checkpoints is damaged detected?
G1, S, and G2
What happens if DNA damage is detected at the cell cycle checkpoints?
ATM and ATR kinases are activated rapidly by phosphorylation. These transmit signal to proteins (eg ChK1, ChK2, p53) which activate other proteins that cause cell cycle arrest
Which proteins detect damage at cell cycle checkpoints?
ATM and ATR kinases
Why is arresting the cell cycle a good idea when DNA damage is detected?
More time allowed for DNA repair and activation of apoptotic pathway if damage is too great to be reapired
Which protein pathway fixes bulky adducts and pyramadinie dimers?
Nucleotide excision repair
What type of DNA damage is fixed by base excision repair?
Abasic site single-strand breaks
Which repair pathway fixes base pair mismatch?
Mismatch repair, MMR
What type of damage is repaired by homologous recombination repair, HRR, or non-homologous end-joining, NHEJ?
Double-strand breaks and interstrand crosslinks
Which repair pathway fixes guanine alkylation?
Methyl-guanine methyl-transferase pathways, MGMT
Describe what happens when DNA polymerase makes a mistake
DNA polymerase will make a mistake every 1/100,000 nucleotides
The wrong nucleotide is inserted, causing insertion/deletion mutations
Proof-reading corrects majority of mistakes, however mismatch repair is also used
The wrong base is removed and DNA extension is attempted again with correct base
Describe the sequence of events involved in mismatch repair
MutS protein binds to mismatched base and recruits MutL protein & endonuclease which degrees faulty strand past mismatch
PCNA helps direct action
RPA binds to single strand and is copied by replicative polymerase.
Strands are sealed by ligaments.
List the proteins involved in mismatch repair
MutS MutL Exonuclease PCNA (proliferating cell nuclear antigen) RPA (replicative protein A) Replicative polymerase Ligase
Describe the action of direct reversal repair using methylated guanine as an example
Guanine is methylated on the O6 position (=> O6methylguanine)
MGMT accepts methyl group which prevents O6MeG from pairing with thymine instead of cytosine in replication
List the five core steps of base excision repair in order
- Excision of base
- Incision of strand
- Processing of ends
- Repair synthesis
[- gap filling and ligation]
Describe how base excision repair is used to repair oxidative damage to bases
Damage base is removed by DNA glycosylase
Excision of base results in an AP site (apurinic or apyrimidinic)
Incised by AP endonuclease (APE1) to create single strand break
PARP1 binds to break (creating intermediate) and helps to recruit other DNA repair factors
DNA polymerase beta adds correct complementary base and DNA ligase III seals end
What happens if ends are not processed and are resistant during base excision repair?
DNA polymerase adds a run of nucleotides between 2-6 and FEN1/PCNA excises flap structure
DNA ligase I seals ends
What are the differences between short patch and long patch base excision repair?
Short patch uses ligase III to seal ends ad long patch uses ligase I
Long patch requires DNA polymerase to add a run of nucleotides and FEN1/PCNA to excise flap structure.
Ends can be processed in short patch but not long patch
LIst the proteins involved in base excision repair (both long and short patch)
DNA glycosylase AP endonuclease (APE1) PARP1 Polymerase beta DNA ligase III [short patch] DNA polymerase [long patch] FEN1 [long patch] PCNA [long patch] DNA ligase I [long patch]
As well as oxidative damage to bases, what else can base excision repair be used to repair?
AP sites and strand breaks
What are the two types of nucleotide excision repair and what is the difference?
Transcription coupled NER: repairs lesions on coding strand
Global genome NER: repairs rest of genome
There are differences in initial step of pathway.
Describe the nucleotide excision repair pathway
Lesion is recognised by CSA and CSB in transcription coupled NER and is recognised by XPE, RAD23B and DPB in global genome NER.
TFIIH removes single stranded DNA segment which contains damage (via XPG and ERCC1XPF)
DNA polymerase uses undamaged DNA for template to replace excised strand, PCNA often acts as a cofactor for polymerase.
Ligase III joins DNA strands together
Why are double stranded break repairs considered the most important repair pathway?
There is a risk of deleting genetic information if it is not repaired
What are the two most common pathways which fix double strand breaks?
Homologous recombination (HR) Non-homologous end-joining (NHEJ)
Describe the pathway of non-homologous end-joining
Ku recognizes and binds to DNA ends, and then recruits subunit DNA protein kinase (PKcs)
Ends are processed via nucleuses (eg Artemis) OR DNA nucleotides are added to make ends compatible
XRCC4, Ligase IV, and XLF complex seals break
Why is NHEJ considered error prone?
Small additions/deletions may occur
Describe how homologous repair is used to fix double stranded breaks
During DNA replication, sister chromatids are used as a template
MRN CTIIP complex generate single strand of DNA which replication protein A (RPA) binds to
RAD51 nucleoprotein filaments search or identical sequence on homologous template, invades template, and uses correct sequence
The DNA is copied and used to mend strand, repairs are completed by joining strands
What are the differences between NHEJ and HR
HR uses identical sequence from sister chromatids
NHEJ occurs throughout ll of cell cycle
HR only occurs in S and G2 phases
Why is the balance o DNA repair important?
Too little repair and mutations accumulate which cause genetic instability
Too much repair and the apoptotic pathway is inhibited which may enable cells with badly damaged DNA to attempt to repair, but instead misrepair and survive
What are 2 examples of real life situations we know cause DNA damage and cancer?
- UV light & skin cancer: pyramidne bases absorb UV light making them more reactiv towards other bases, forming dimers (repaired by nucleotide excision pathway)
- Smoking & lung cancer: carcinogens cause bulky adducts that bind to DNA, also contains free radicals causing oxidative damage. P53 mutations in lung cancer
What is an example of a bacterial infection that causes cancer?
H Pylori, associated with stomach cancer.
Causes chronic inflammation which increases Reactive Oxidative Species, causing DNA damage
What is an example of a viral infection that causes cancer?
Hepatitis B and C
Causes chronic inflammation which increases ROSs, causing DNA damage
What % of cancers are associated with infections?
~10-15%
What is functional redundancy?
The idea that if the main pathway for a type of DNA damage doesn’t repair the damage, a second pathway i usually available to act as backup mechanism
List some syndromes associated itch germline mutations in DNA damage
Li Fraumeni Cockayne’s Xeroderma Pigemtosum Franconi’s Hereditary breast/ ovarian cancer Hereditary no polyposis colorectal cancer Nijemegan breakage Altaxia talagiactasia Bloom, Werner, Rotham, Thompson
What can genetic instability result in?
Mutations
Tumour progression
Pathogenesis
Resistance to treatment
What are mutations?
Permanent changes to DNA sequence
What causes mutations?
Damage to DNA
Errors in replication
What are the possible effects of mutation?
Prevent proteins from being expressed
Change function of proteins
Cause over-expression/activation of protein
What are the 4 main forms of mutations?
Single nucleotide variants
Insertions/deletions
Aneuploidy
Microsatellite instability
What is a single nucleotide variant?
A point mutation where one base is changed in an amino acid
What happens if a single nucleotide variant mutation occurs in a DNA binding domain?
Affects p53 confirmation
Downstream signalling is altered
What is the most common single nucleotide variant mutation in cancer?
P53
What is an insertion mutation?
Extra DNA added to gene
Might be to/from a different chromsome
What is a deletion mutation?
DNA removed from gene, entire gene is missing, or chromosome breaks and information is lost
What may insertion/deletion mutations result in?
amplification of oncogenes
Deletion of TSGs
What is an inversion mutation?
Where a chromosome breaks in two places, flips the piece, and re-inserts it back into the chromosome
What is a translocation mutation?
Where a piece of chromosome breaks off and attaches to the end of chromosome
What is an example of a translocation mutation and what part of the chromosome(s) does it affect?
chronic myeloid leukemia (CML)
Section of chromosome 9 containing ABL breaks and attaches to chromosome 22, creating product BCR-ABL (which is an oncogenic tyrosine kinase)
What is Aneuploidy?
The addition/deletion of whole chromosomes
What happens if the mitosis checkpoints that ensure chromosome segregation goes wrong?
Misregulation of chromosomes
Abnormal number of chromosomes proudiced in daughter cells
May promote cell growth / tumour genesis
What are microsatellites?
Repeated DNA sequences spread throughout genome
Generally, how many base pairs make up a microsatellite?
Around 1-6
What is a genetic fingerprint?
The length and locus of an individual’s microsatellites
What is microsatellite instability?
When microsatellites are not coherent within the individual
What causes microsatelite instability?
The repeating units cause slipping during replication, resulting in insertions/deletions
What is an example of a cancer caused by microsatellite instability?
Colorectal cancer
What advantages do mutations have fr cancer cells?
Increase proliferation
Decrease apoptosis
Result in genetic instability
Affect all hallmarks of cancer
Why are certain parts of genome more mutation prone?
Different genes have different mutation rates
What are some examples of genes mutated in cancer?
P53
Rb (a TSG)
Ras and EGFR (oncogenes)
What is a ‘caretaker’ gene?
A first mutation in a DNA damage and response gene which results in further mutations occurring rapidly
Describe intra-tumour heterogeneity
A tumour with different mutations within in it
One part of the tumour may be different to another part depending on the mutations that occur there
