2. DNA Damage and Repair

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

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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 

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Keto/enol: Mispairing of Tautomers

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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 ​​

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

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Replication Errors:Base Slipping

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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

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  

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

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Spontaneous Lesions: Deamination

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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

Spontaneous Lesions: Deamination

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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

Spontaneous Lesions: Oxidation of Bases

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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

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

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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

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Induced Lesions(Mutagens): Bulky Adducts

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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

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)

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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)​​​​​​​

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

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 

Mutagens Cause Arrest of Fork: Pol V

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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

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Context: Ames Test

Context: Consequence of Blocked Fork

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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

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

Context: Forward Genetic Screens

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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  

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Zebrafish Screening

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Mutations Eukaryotes vs Bacteria

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Pie de foto: : Bacteria happy to let mutations accumulate

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)

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   

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

Photoreactivation

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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

Excision Repair: Base Excision Repair (BER)

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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

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)

Excision Repair:Nucleotide Excision (NER)

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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

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

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Excision Repair: Mismatch Repair (MMR)

In eukaryotes dont need MutH as strand discrimination is not based on methylation  Have MutL and MutH

DSB Repair: Non-homologous endjoining

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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

DSB Repair:Homologous Recombination(HR

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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

Fork Arrest: Damage In Front of Fork

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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

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

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 

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Holiday Junction

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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 

2. Fork regression, HJ cleavage, BIR

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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)

2. Fork regression, HJ cleavage, BIR

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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

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  

3. Regression, free 3', invasion, resolution HJ

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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 

Context:Exploit NHEJ to make Mutations

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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

Context:Exploit HR to make Mutations

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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 

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2. Fork regression, HJ cleavage, BIR

How DNA is Damaged

Nucleotide decay Depurination Depyrimidination Spontaneous deamination Endogenous and exogenous agents Oxidative damage Chemical exposure Radiation (X-rays)  

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

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

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Cancer: Loss of Repair Functions

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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 

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

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

Mutation Signatures

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ds Breaks: Triggers

Reactive oxygen species/ ionising radiation  DNA topoisomerase inhibitors (chemotherapies) Replication stress

ds Breaks: Repair via HR

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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 

ds Breaks: NHEJ Repair

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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

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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 

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

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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 

DNA Damage Response in Cancer

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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 

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

P53 and Mutation Signatures (G-T)

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Pie de foto: : G-T transitions occur in p53 gene

P53 and Mutation Signatures (G-T)

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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

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   

Therapeutic Strategies

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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   

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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

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