17992280
2. DNA Damage and Repair
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Criado por Chloe Cavarretta
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Resumo de Recurso
Slide 1
Learning Outcomes: L5-7
Understand that there is a variety of mechanisms for both spontaneous and induced errors to be introduced in to the DNA sequence and be able to provide examples of them
Explain how DNA damage can affect the replication fork machinery during DNA replication
Discuss some uses of mutagenic compound in biology
Describe the Ames test and its use in screening for mutagenic compounds and the uses of mutagenic compounds in forward genetic screens
Understand that there is a variety of mechanisms for the repair of DNA damage and that these mechanisms deal with distinct types of damage
Explain how the replication fork machinery can recover from stalling or collapsing after encountering DNA damage during replication
Summarise how NHEJ and HR can be exploited by using the CRISPR-Cas9 system
Appreciate the consequences of DNA damage
Understand how DNA repair is integrated with the cell cycle
Be able to outline how hypermutable cell lineages emerge
See how this is related to human cancer
Slide 2
Slide 3
Replication Errors: Keto/enol
Major source of errors: Keto/enol equilibrium
DNA bases exist as 2 tautomers
Rapidly interconverted constitutional isomers (same chemical formula, different atom arrangement)
Distinguished by different bonding location of labile H atom and different location of double bond
Rapid equilibrium
DNA bases strongly favour keto isomer
Slide 4
Keto/enol: Mispairing of Tautomers
Differences in base pairing of tautomers:
If T flips from keto to enol:
3 H bond donor instead of 2, T can bp with G
Or if T keto and G flips to enol
Same principle for A and C but amino/imino rather than keto/enol
If A or C are imino- A and C can bp
Slide 5
How Mispairing Affects DNA Replication
A-T results in G-C or G-C results in A-T:
A-T(keto) -> A-T(enol)
Round of replication: A-T + G-T(enol) -> G-T(keto)
T enol bp with G causing mismatch
Temporarily in enol (keto preferred) so flips back
T-G could either be repaired or go through another round of replication
Round of replication(of G-Tketo): G-C and A-T
Slide 6
Replication Errors:Base Slipping
Cause small deletions/insertions
DNA backbone has some give when replication fork passing
Loops can form= new strand and template different lengths:
Slippage in new strand= +1 addition
Slippage in template= -1 deletion (of new strand)
Issues occur at next round of replication
Slide 7
Base Slipping: Small Deletions/Insertions
Can happen on a larger scale:
Indels= Insertions and deletions
Tandem repeats= repeated short sequences of bp
Difficult to get accurate replication:
Polymerase moves along repeats, may skip along a repeat= deletions
Polymerase might slip back on repeat and carry on= addition
Slide 8
Spontaneous Lesions: Loss of Bases
Loss of base by hydrolysis forming Apurinic or Apyrimidinic sites= AP sites
AP site dont have base pair= gap in backbone
Like in mismatches AP sites also recognised by repair mechanisms
If not repaired- missing info on template DNA so get random incorporation of base (mainly A)
A bp with T so GC-> TA mutation
Slide 9
Spontaneous Lesions: Deamination
Amino group of cytosine can be deaminated= uracil formed
Uracil used in RNA, not DNA
Usually noticed by repair mechanisms and cut out
5MeC is used gene regulation in eukaryotes (CpG islands) and DNA licencing in prokaryotes
Deamination of amino group of 5MeC= T formed
Slide 10
Spontaneous Lesions: Deamination
5MeC mutational hotspots in lacl genes in E.coli:
Methylation only happens at particular motifs, not to every C
Plot position of methylated C and compare to normal C
Frequency of GC-> AT mutation higher at places where 5MeC present
Slide 11
Spontaneous Lesions: Oxidation of Bases
Oxidation of G= additional H bond donor in a different place, G can bp with A
To bp with A has to rotate, contort backbone but strong H bond donor overcomes
Oxidation of G is frequent
Caused by ROS eg H2O2 (normal products of metabolism but increased by radiation)
Rad= more ox= more mutants
Slide 12
Spontaneous Lesions: Oxidation of Bases
How 8-oxo-G causes mutations:
If not repaired and replication occurs: Form oxo-G-A strand and G-C strand
Can either repair or go through another round
Another round of replication: another oxo-G-A strand and A-T
GC-> TA or AT-> GC
Slide 13
Induced Lesions(Mutagens): Alkylation
Mutation by alkylating agents (EMS and ENU)
Add alkyl group onto base
EMS transfers ethyl group to 6' position of G
G fixed in enol form= bp with T
Causes GC->AT transitions
Slide 14
Induced Lesions(Mutagens): Bulky Adducts
Base damage by bulky adducts
Alfatoxin and benzopyramine dont react with DNA themselves but metabolised into mutagens which reacts strongly with DNA bases-> generate base pair adducts
Adducts prevent replication fork being able to pass through= replication arrest and strands break
Slide 15
Induced Lesions(Mutagens): UV
UV=mutagen, cause cross-linking
Crosslinks adjacent pyrimidines to each other= cyclobutyl ring (prymidine dimer) and reactive 6-4 phosphoproduct
Changes shape of backbone- distorts
Replication fork cant pass distortions, arrests fork (similar to adducts)
Slide 16
Induced Lesions(Mutagens): Intercalating
Intercalating agents: Proflavin, Ethidium
Are used in biological applications- add to gel and cause fluoresence when intercalate, can stain NA
Planar molecules that slip between bps and shift distance between bps
Can happen during DNA replication= errors, DNA pol passes trhough agent and cause strand slippage
Frequent deletions and insertions (usually large scale)
Slide 17
Mutagens Cause Arrest of Fork
Bulky base modifications and UV generated photoproducts inhibit DNA replication and transcription so are toxic
Also lead to mutations due to use of innacurate bypass polymerases
Slide 18
Mutagens Cause Arrest of Fork
Pol V bypass polymerase, can bypass lesions at stalled replication forks:
If fork stalls, pol III stalls at site of damage but cell wants to keep copying DNA
Pol V replaces pol III and carries on replication, pol V:
Lacks proofreading ability
Sloppier copier
Error-prone DNA synthesis
Higher rate of mutations
Slide 19
Mutagens Cause Arrest of Fork: Pol V
Slide 20
Context: Ames Test
Test for carcinogenicity/mutagenicity, takes into account that some compounds need to be metabolised to be mutagenic, no need to use animals
1. Incubate compound X (alfatoxin/benzopyrine) and liver extract
2. On plate in Salmonella mutant for genes in His synthesis (no histidine made my bacteria and none in medium so shouldnt grow)
3. Put paper disc soaked with compound X on plate- if put disk on and get colonies indicates you have revertants
Control= Colonies on top plate formed by spontaneous mutation able to produce His
Bottom plate(middle): conc of mutagen high, DNA severely damaged by too many mutations so no growth
Bottom plate(outside): Rate of reversion, bacteria can synthesise His again
Mutagen has caused another mutation which counteracts original one and produces functional protein, size of halo determines how powerful mutagen is
Slide 21
Context: Ames Test
Slide 22
Context: Consequence of Blocked Fork
Mutation in leading strand synthesis more likely to cause arrest:
DNA damage in lagging not a barrier to fork progression as replication done by repeated annealing of RNA primer
If damage caused gap, re-prime and filling of gap as normal
Leading: DNA pol wont progress past gap, re-priming is rare
Slide 23
Context: Forward Genetic Screens
Forward:
1. Mutagenize random amount of genes- use ENU/EMS
2. Screen animal mutations that youre interested in
3. Isolate different mutant phenotypes
4. Cloning- find out what gene is causing the phenotype
Reverse:
1. Start with gene you know are implicated in pathways
2. Functional analysis- what happens when gene/gene product removed
3. What mutation occurs
Slide 24
Context: Forward Genetic Screens
Slide 25
Context: Forward Genetic Screens
Eg. Zebra fish: Male fish bathed in small amount of ENU and mutations introduced as sperm produced (finesse conc of ENU to get 1 or 2 per sperm)
Cross mutant sperm and WT egg
Progeny= F1 heterozygous fish (heterozygous for many mutations)
Outcross heterozygous F1 with WT- to reduce number of mutations per chromosome due to chromosome crossing over
Progeny= F2 50% heterosygous for few mutations, 50% WT
Incross F2 with F2- try to get specific mutations
Progeny= F3 25% WT, 25% homozygous mutant, 50% heterozygous mutant
Slide 26
Zebrafish Screening
Slide 27
Slide 28
Mutations Eukaryotes vs Bacteria
Rubrica: : Bacteria happy to let mutations accumulate
Slide 29
How DNA Damage Occurs
Spontaneous mutations:
Replication errors- base changes, deletions, insertions)
Spontneous lesions- (Loss of bases, deamination, oxidation)
Induced mutations:
Alteration of bases (base change)
Intercalating agents ( deletion/insertion)
UV radiation (pyrimidine dimers, UV phosphoproducts)
Ionising radiation (strand breakage, loss of bases, oxidation)
Slide 30
Damage Prevention
Proofreading by DNA pol:
Normal DNA Pol- Can sense mismatch, go backwards and remove wrong base via endonuclease activity to minimise mismatches
In emergency may need to use Pols that dont have proofreading activity- better to have inaccurate replication rather than none?
Hydrolysis of damaged nucleotides:
Eg. Ecoli 8-oxo-GTP detected as damaged nucleotide
Hydrolysed by MutT to 8-oxo-GMP
8-oxo-GMP cannot be used in replication-> preventing DNA damage
Slide 31
Direct Reversal of Damage
Alkyltransferases:
Remove methyl groups via enzymatic hydrolysis- and methylate own aa in AS instead
O6-methylguanine-DNA methyltransferase, MGMT, removes methyl group
Photoreactivation:
Reversal of pyrimidine dimers (photodimers) after UV damage
Photolyases activated by low energy light and remove crosslinked bonds
Slide 32
Photoreactivation
Slide 33
Excision Repair: Base Excision Repair (BER)
For small DNA lesions (single bp)- base taken out of DNA backbone but it remains intact
DNA glycosylase- different ones for specific bases eg 8-oxo-G, DNA glycosylase OGG1
Precise
Slide 34
Excision Repair: Base Excision Repair (BER)
1. Offending base removed by DNA glycosylase, leaving AP site
2. AP site cleaved by AP endonuclease
3. AP lyase removes deoxyribosephosphate
4. DNA pol fills gap with correct nucleotide
5. DNA ligase seals ends
Slide 35
Excision Repair:Nucleotide Excision (NER)
Nucleotide excision repair (NER)
For slightly larger scale damage (bulky adducts)
Excises oligonucleotides rather than single base
A big DNA repair complex (includes helicase)
Slide 36
Excision Repair:Nucleotide Excision (NER)
1. DNA repair complex recognises base damage
2. Incision of DNA strand on either side
3. Removal of oligonucleotide by complex
4. DNA polymerase fills gap
5. DNA ligase joins ends
Slide 37
Excision Repair: Mismatch Repair (MMR)
Strand specific repair
Bacteria use methylation to discriminate between old and new strands
Newly added wrong bp wont be methylated yet
MutS recognises and binds mismatch
MutL and MutH bind, conformational change in backbone to pull DNA through complex
MutH (endonuclease) cuts new strand DNA only before GATC consensus (point of methylation)
DNA polymerase fills gap in single strand
Slide 38
Excision Repair: Mismatch Repair (MMR)
In eukaryotes dont need MutH as strand discrimination is not based on methylation
Have MutL and MutH
Slide 39
DSB Repair: Non-homologous endjoining
Double stranded break repaired by NHEJ
Not accurate- can lead to insertions/deletions (mainly deletions)
DNA ends bound by complex with endonuclease activity
Complex trims ends (overhangs)= cause of insertions/deletions
Joining of ends
Slide 40
DSB Repair:Homologous Recombination(HR
Accurate repair
Occurs after G1- synthesis dependent strand annealing, relies on intact sister chromatid or another intact DNA strand to act as template
Other DNA acts as template, formation of holiday junction
Annealing to strand on other DNA
Polymerase attaches and synthesises rest of each strand
Junction hydrolysed, end up with 2 intact bits of DNA
Slide 41
Fork Arrest: Damage In Front of Fork
Damage in lagging strand template is not a barrier to replication fork progression
Re-priming and okazaki fragment formation
Damage in leading strand leads to fork arrest
Re-priming is rare
Slide 42
Fork Arrest: Continuing DNA synthesis
When leading strand stops, lagging strand cant continue much longer- fork arrest
Three ways to recover:
Fork regression, template switch, reversal
Fork regression, HJ cleavage, BIR
Fork regression, producing free 3' end of extruded DNA, invasion of DNA on other side, resolution of dHJ
Slide 43
1. Fork regression, template switch, reversal
Allows replication to continue with or without repair
Lesion in DNA on lagging strand and stops, lagging continues
Regression: Fork regresses back and get extrusion of lagging strand
Template switch: lagging used as template for the leading
Reversal and DNA repaired, newly synthesised DNA is annealed across newly repaired DNA
OR Reversal and lesion is bypassed newly synthesised DNA is annealed across site of damage
Slide 44
Holiday Junction
Replication fork regression produces holiday junction
3 different ways of showing holiday junction
Strand from one bit of DNA invades another strand- cause cross over
Slide 45
2. Fork regression, HJ cleavage, BIR
Regression: Leading and lagging extruded out of back of fork to form holiday junction
Holiday junction cleaved at stalled fork which produces an intact template and a broken ended DNA molecule (red circle)
Slide 46
2. Fork regression, HJ cleavage, BIR
Replication restart by homologous recombination: Break Induced Recombination
Loose end chewed back by exonuclease leaving overhanging end (5' to 3')
In bacteria, RecA binds to ssDNA
Homology search for template strand
Strand invasion of template causes D loop formation
Re-establishment of replication fork
End up with little loose end, strand primed with RNA primer to fill in
Formation of another HJ to resolve existing HJ
Slide 47
3. Regression, free 3', invasion, resolution HJ
Single-ended DSBs pose a risk: in last mechanism, search for homologue required which might not be accurate eg if their is repetitive DNA
Safer alternative= process extruded DNA to create a larger region of homology and invade strand in front of fork
Single ended DSBs prevented
Lower risk of ectopic recombination
Slide 48
3. Regression, free 3', invasion, resolution HJ
Fork regresses and extrusion of DNA out of back to get dsDNA
Chew off end to get 3' tail
Get bigger region of homology and invade strand
Form bigger and more HJs
Cleavage of dHJ (double)
Restart replication fork
Slide 49
Context:Exploit NHEJ to make Mutations
Using CRISPR-Cas9 system
Guide RNA (gRNA) and Cas9 proteins introduced into cell-> Cas9-gRNA complex
gRNA binds complementary DNA and Cas9 cleaves DNA to get ds break
Cell recognises cleavage and NHEJ repair
Inaccuracy of NHEJ= insertion, deletions, frameshifts
gRNA targets exon in an early gene to hopefully get frameshift by cleavage
Form a zoo of mutations but want a frameshift
Slide 50
Context:Exploit HR to make Mutations
guide RNA-Cas9 complex causes ds break
Introduce template with big homologous region to target strand (but middle part is bit to insert)
At a certain rate, homologous arms bind broken ds and polymerase inserts sequences of interest
Slide 51
2. Fork regression, HJ cleavage, BIR
Slide 52
How DNA is Damaged
Nucleotide decay
Depurination
Depyrimidination
Spontaneous deamination
Endogenous and exogenous agents
Oxidative damage
Chemical exposure
Radiation (X-rays)
Slide 53
Mismatch Repair
DNA polymerase incorporates wrong bp 1 in 10^5
Proofreading by polymerase= reduces error rate to 1 in 10^7
Mismatch repair= reduces error rate to 1 in 10^9
Slide 54
Mismatch Repair
Mismatch Repair
Detect distortions when wrong base pair added
By proteins MSH2 and MLH1 that are coupled to replication fork
Intervening bp's removed and resynthesised to fill the gap
Mismatch repair genes MSH2 and MLH1 often mutated in cancer cells
Slide 55
Cancer: Loss of Repair Functions
Loss of repair functions is an early event in cancers:
Staining for MHL1 (black nuclei with MHL1 and blue without)
Tumour tissue T has no MHL1 but neither does surrounding normal tissue
Suggests that loss of MHL1 is an early event that predates other tumorigenic changes
Slide 56
Mismatch Repair Defects
Short sequence repeats (SSR) are prone to instability
Genes with these repeats are reliant on mismatch repair - more sensitive to loss of mismatch repair
TGF-b receptor functions in G1 to respond to antigrowth signals:
Instability of TGF-b receptor due to SSR repeats results in failure to respond to antiproliferation signals= hallmark of cancer
Slide 57
Mutation Signatures
Some mutation signatures include: repeat instability, G-T transitions, C-T transversions, Deletions
Signatures are classified according to the type of tumour in which they emerge
eg Signature 1 (next slide) is in all tumour types and is a consequence of spontaneous deamination
Signature 4 is a consequence of polycyclic aromatic hydrocarbons (cigarette smoke)
Some mutation signatures are associated with specific mutagen and they often reflect where the mutagen enters the body;
Polycyclic aromatic hydrocarbons (PAH)- lung epithelium
UV- epidermis
Aflatoxin- liver
Slide 58
Mutation Signatures
Slide 59
ds Breaks: Triggers
Reactive oxygen species/ ionising radiation
DNA topoisomerase inhibitors (chemotherapies)
Replication stress
Slide 60
ds Breaks: Repair via HR
Melting chromatid region where ds break is on helix
Invasion by the free end of the severed DNA strand into template (other chromatid)
Extension of the severed strand by DNA pol
Re-joining of severed strands
Error free because all information is provided by other chromatid
Restricted in cell cycle as other chromatid is not yet formed in G1
BRAC1 and BRAC2 proteins involved in breast cancer, BRAC1 involved in assembling proteins that repair ds breaks
Slide 61
ds Breaks: NHEJ Repair
Enzymes detect and bind free end of DNA and catalyse ligation
No need for extensive sequence homology- need short region of homology to bring ends together
Error prone= involves loss of sequence
Repairs DSB in all phases
Slide 62
Checkpoints
DNA damage response is the cells ability to detect damage and arrest the cell cycle
Can feed into either passage through:
Restriction point R- entrance to S blocked if genome is damaged
S phase- DNA synthesis blocked if genome damaged
Mitosis check points:
Entrance to M blocked if DNA replication not completed
Anaphase is blocked if chromosomes are not assembled on the mitotic spindle
Checkpoints often disabled in cancer cells
Slide 63
Proteins involved in DDR:
Sensors- detect presence of structure abnormalities in DNA
Transducers- respond to sensors and transmit signal throughout nucleus
Effectors- switched on as a consequence of transducers, provide wide range of responses mediated through effectors
DNA Damage Response
Slide 64
DNA Damage Response in Cancer
Mutations to proteins involved in surveillance and repair are implicated in tumours:
eg surveillance- checkpoint kinase implicated in lymphomas and breast cancer, chk2 mutations occur in early development of tumours
eg repair- mismatch repair in colon cancer
Slide 65
DNA Damage Response in Cancer
Chk2- Checkpoint kinase 2
Arrests the cell cycle and activates checkpoint so cell can repair damage
Total Chk2: present in normal cells, down-regulated in breast cancer so cell cant arrest cycle
Activated Chk2: in normal cells not activated, in breast cancer cells is activated
Slide 66
DNA Damage Response in Cancer
p53 gene product is a tumour suppressor that is present in low amounts in cytoplasm in undamaged DNA and activated via signal in damaged DNA and transcribed in nucleus
Mutations in p53 gene or its regulation are the most common changes in cancer. Activated p53 controls the decision to:
Divide- ignore damage and enter S phase
Arrest- pause cycle and repair damage
Die- apoptosis if damage is too severe
Role of p53:
Promotes expression of Cdki (cyclin dependent kinase inhibitor)- p21
p21 inhibits cyclin E and A/Cdk- preventing entry to S
p21 inhibits PCNA - halts ongoing respiration
Slide 67
P53 and Mutation Signatures (G-T)
Rubrica: : G-T transitions occur in p53 gene
Slide 68
P53 and Mutation Signatures (G-T)
Spectrum of mutations detected in p53 gene in lung tumours
Orange= G-T transition caused by PAH
Percentage of base changes that is G-T:
In all cancers= 15%
In lung tumours in non-smokers= 21%
In lung tumours in smokers= 33%
Smoky coal= 75%
The type of mutation provides clues about the mutagenic agent
The location of common mutations in p53 gives clues about the functional sites in the protein
Slide 69
Therapeutic Strategies
Exploiting knowledge of the p53 gene:
p53 deficient cells are more reliant on other DDR pathways so more susceptible to inhibitors - which allows the sensitization of tumours to treatments
A cancer cell with compromised p53 and an inhibited DDR response will continue to divide in the presence of the damage but damage such as ds break prevents the cell cant go through mitosis
Slide 70
Therapeutic Strategies
Slide 71
Loss of DDR is an Early Event in Cancer
Precursor lesions (population of cells beginning to form tumour) of breast, lung, colon, bladder cancers express markers of an activated DDR
Urothelial precurosr lesions have elevated expression of activated chk2 compared to normal tissue
Slide 72
Loss of DDR is an Early Event in Cancer
Hypothesis:
At an early stage cells experience oncogenic stress (small constant level of internal stress)
They respond by activating protective pathways- DDR(arrest) or die
Events that compromised these pathways (eg mutation that affects arrest) allow cell proliferation and survival
Daughter cells have defective checkpoints and instability enabling rapid tumour progression
Chronic activation-> deactivation-> hypermutable population
Slide 73
Hallmarks of Cancers
Common acquired traits:
Self-sufficiency in growth signals
Insensitivity to growth-inhibitory signals
Tissue invasion and metastasis capability
Limitless replicative potential
Sustained angiogenesis
Evasion of apoptosis
Reprogramming energy metabolism
Evasion of immune system
Enabling events:
Loss of genome surveillance and checkpoint control
Destabilisation of nucelar organisation
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