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MBB313 - Genome Stability and Genetic Change > El-Khamisy - SSBR > Flashcards

El-Khamisy - SSBR Flashcards

1
Q

Can kinases be targeted?

A
  • yes, v druggable targets

- biggest category of protein targets for inhibitors which have progressed to clinical trials

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2
Q

Is DNA stable?

A
  • must v stable as can find it in ancient bodies
  • but only stable if in body
  • damage to DNA is mainly hydrolysis of water –> gen reactive species
  • approx 7 breaks per cell per min
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3
Q

Are DNA breaks good or bad?

A
  • generally bad, as hallmark of cancer
  • but can be good (important that engineering these breaks, ie. they are controlled)
  • -> Ig diversity (req engineering of DNA break)
  • -> meiosis
  • -> induce gene exp if in promoters in neurones of NS
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4
Q

What are the consequences of unrepaired DNA damage?

A
  • cell death (if stop replicating) –> causes degen disease, autoinflam
  • cell survival –> causes cancer
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5
Q

How is genome stability achieved?

A
  • genome packing (spatial organisation) –> chroms in certain location in nucleus are more privileged in terms of fixing
  • geometry (shape and size)
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6
Q

How does genome instability contrib to cancer?

A
  • enrichment of prot coding mutations in DDR (DNA damage response) genes
  • age is most common predisposing factor in cancer –> as much more likely to have accum mutations in prot coding genes by this time
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7
Q

What happens if DNA breaks in non-replicating cells of NS?

A
  • always causes cell death –> causes neurodegenerative disorders
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8
Q

Why are topoisomerases important?

A
  • DNA 2-3m –> packaged and compacted into nucleus
  • gens lots of knots/entanglements –> problem as need to unwind
  • done by tpms
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9
Q

What are the diff challenges to genomic integrity?

A
  • oxidative and prot linked DNA breaks
  • DNA rep
  • gene transcrip
  • ribose contam
  • spont base loss
  • endogenous base mods
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10
Q

How are oxidative and prot linked DNA breaks a challenge to genomic integrity?

A
  • DNA topoisomerases sometimes don’t reseal break and left w/ prot attached covalently to DNA, will block transcrip/rep/recomb etc. –> can happen if in prox to ROS
  • formaldehyde used to fix cells etc. in lab –> can cross link w/ DNA
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11
Q

How do topoisomerases carry out their role?

A
  • make break in 1/2 strands, allows swivelling of other strand or duplex, then reseal
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12
Q

What is formaldehyde?

A
  • byproduct of demethylation of histones and DNA
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13
Q

How is DNA rep a challenge to genomic integrity

A
  • has to rep every time cell divides

- can make mistakes, leading to DNA damage

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14
Q

How is gene transcrip a challenge to genomic integrity?

A
  • as RNA pol travels across DNA to make RNA, gen +vely wound DNA in front of pol and behind is -vely supercoiled
  • this is nascent RNA, so can pair w/ duplex DNA –> gen 3 strand nucleic acid structure = R-loops
  • can be bad as expose 1 DNA strand, so can be easily attached (not protected anymore), so considered major source of DNA instability
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15
Q

What are R loops made up of?

A
  • DNA-RNA hybrids
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16
Q

How can R loops be seen in the lab?

A
  • w/ Abs to visualise cells
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17
Q

What seqs can favour R loop formation, and why?

A
  • repetitive regions and GC rich seqs favour R loop formation
  • as any event that slows down pol increase chance of formation
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18
Q

Where are repetitive and GC rich seqs often found?

A
  • termination point –> may be good as helps termination signals and causes pol to fall off
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19
Q

What physiological and pathological sources are there of transcrip byproducts?

A
  • physiological = eg. rDNA seqs, CFS

- pathological = eg. nt repeat expansions (in motor neurone disease, Huntington’s etc.)

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20
Q

What are the physiological and pathological consequences of R-loops?

A
  • can be useful to guide termination of transcrip
  • can relieve topological constraints and supercoiling
  • major source of DNA breaks if left unresolved
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21
Q

How is ribose contam a challenge to genomic integrity?

A
  • RNA much more unstable as ribose, not deoxyribose

- presence of OH makes bond much more unstable (as labile), so contam makes DNA unstable

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22
Q

What is the only diff between ribose and deoxyribose?

A
  • is H/OH on carbon 2
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23
Q

What is the source of ribose contam to DNA?

A
  • take ribose by mistake, instead of deoxyribose, as so abundant (at least 2x order of magnitude)
  • DIAG*
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24
Q

How is spont base loss a challenge to genomic integrity and how common is it?

A
  • labile under physiological conditions (hydrolysis)

- v common event

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25
Q

How is endogenous base mods a challenge to genomic integrity?

A
  • gens another chemistry, which may mispair and cause damage
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26
Q

What would happen if mod bases not repaired?

A
  • deaminated in alkaline conditions
  • if change chem of nt, then will NOT pair w/ canonical bp
  • if remove amine group from C, get U, if try to rep U end up with mutation from GC to AT –> if happens to be oncogene etc. then big consequences
  • also other endogenous deamination reactions
  • -> eg. deamination of meC is major cause of mutation in human cancers, eg. p53 mutations
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27
Q

How is oxidation a problem as a base mod?

A
  • ROS are byproducts of normal aerobic metabolism
  • over 80 diff products known, react w/ double bonds, prod ss breaks and lipid peroxidation
  • if base oxidised then mispair
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28
Q

What endogenous threats to DNA are there?

A
  • rep
  • transcrip
  • ribose contam
  • reaction w/ mols in cell, such as water and O
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29
Q

What exogenous threats to DNA are there?

A
  • reaction w/ mols outside cell
  • UV
  • cosmic rays
  • man made chemicals
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30
Q

What type of breaks are more common?

A
  • SSB more common than DSB
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31
Q

What are the main steps in SSB repair?

A
  • damage detection –> by PARP, makes poly ADP-ribose polymers, mark break site so cell can fix it
  • end processing –> to ligate 2 ends together 3’ must be hydroxyl and 5’ phosphate (in breaks this often not the case), so need trimming to restore them back to this (diff enzs dep on what specific change is)
  • gap filling –> often enz cuts a bit further along so lose some nts, so need pol to prod flap of DNA
  • ligation → DNA ligase to seal remaining nick
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32
Q

How would you study process of SSB?

A
  • prot interaction w/ key component
  • if pick fundamental step will prob know lot more about whole pathway –> something upstream, eg. XRCC1
  • if pick eg. ligase, or 1 of enzs for end processing only gives specific info about that part of pathway
  • then search for interactions
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33
Q

How do you search for unknown interactions?

A
  • yeast-two hybrid
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34
Q

How does yeast-two hybrid work?

A
  • fuse prot to GAL-4 AD and other prot to GAL-4 DNA BD

- if 2 prots interact will bring 2 doms together and activates promoter, so prod transcript can see in lab

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35
Q

What are the most common reporters used in yeast-two hybrid?

A
  • growth on minimal media = media lacking key component, which is provided by transcript if 2 prots interact, so only growth if interaction
  • or enz activity = colour change if interact
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36
Q

When studying SSBR how was yeast-two hybrid carried out?

A
  • tag XRCC1 w/ BD/AD and have libs, transform yeast w/ lib and construct, plate on minimal plates and look at results
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37
Q

How can you get false +ves in yeast-two hybrid, and how can this be tackled?

A
  • can get transactivating of promoter
  • test w/ -ve control = XRCC1 + lamin
    (-ve control for Y2H always w/ either bait or prey not present)
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38
Q

After carrying out yeast-two hybrid w/ XRCC1 and finding it interacts w/ XIP1, what would be done next?

A
  • already pulled down XIP1 cDNA
  • blast search –> structure of XIP1
  • look at papers on prot –> find role
  • seq alignment of proteins to compare them –> see if have same function
  • found XIP1 aligns perfectly w/ aprataxin –> they are the same gene/prot, ie. that mutated in AOA1
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39
Q

What is the structure of XIP1?

A
  • DIAG*
  • FHA dom = XRCC1 binding
  • NLS dom
  • HIT dom = active site
  • Zn dom = DNA binding
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40
Q

What are the characteristic and symptoms of Ataxia Oculomotor Apraxia-1 (AOA1)

A
  • early onset (1-16 y/o)
  • pathology largely restricted to nervous system (no predisposition to cancer)
  • variable mental retardation
  • ocular motor apraxia
  • cerebellar degen
  • spinocerebellar ataxia
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41
Q

How is aprataxin affected in AOA1?

A
  • mutated

- majority of mutations clustered in HIT (active site) –> means HIT has important function

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42
Q

How can you find the function of a prot?

A
  • culture prot in E. coli
  • clone prot into plasmid (designed to express prots in bacteria)
  • express prot of interest w/ IPTG induction
  • bacteria prod prot, then collect by cell lysis
  • purify by affinity column (prot tagged w/ eg. his, binds nickel well, so only his tagged prot bind nickel beads)
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43
Q

What is the purpose of blasting HIT seq from XIP1?

A
  • see where it aligns w/ other prots

- so find other prots w/ same dom, see role in cell, as may be something similar

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44
Q

How can you determine function of unknown prot?

A
  • radiolabeled DNA (or fluorophore)
  • mix recomb prot w/ substrate and run on denaturing gel
  • tells you its cleaving the AMP
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45
Q

What is the ligation cycle?

A
  • ligase adenylated, so AMP transferred to ligase
  • AMP transferred to 5’ ter and gen AMP-DNA covalently bound
  • free ligase removes AMP and brings 2 ends together –> req hydroxyl
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46
Q

How is the ligation cycle affected when there is a DNA break?

A
  • when DNA break, hydroxyl no longer there, so cannot complete cycle, need enz that cleaves DNA-AMP adducts as blocked here –> resets ligase
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47
Q

What is 1 important source of aborted ligation, and how?

A
  • ribose
  • DNA ligase sees ribose as nick, jumps in trying to ligate it and prod DNA-AMP adduct
  • if no aprataxin to reset ligase, get accum of adducts –> problems w/ cell, affecting NS
48
Q

Is SCAN-1 similar to AOA1?

A
  • yes, cannot diff between these MRIs and those of AOA1
49
Q

What is SCAN-1 and what are the symptoms

A
  • spinocerebellar ataxia w/ axonal neuropathy)
  • pathology largely restricted to nervous system (no predisposition to cancer)
  • variable onset (≈ 15 yrs)
  • cerebellar degen
50
Q

What gene is mutated in SCAN-1, and how is this known?

A
  • TDP1
  • enz w/ phosphodiesterase dom
  • PDD means will phosphorylase bond –> therefore something to do w/ DNA
51
Q

What are abortive TOP1 events, and how can they be resolved?

A
  • any event that causes misalignment of 3’ OH –> eg. presence of nearby breaks, collision w/ transcrip or rep machinery
  • TDP1 cleaves phosphodiester bond between DNA and TOP1
52
Q

How do human and yeast TDP1 differ?

A
  • in yeast mainly works in rep
  • but in humans doesn’t work during rep –> does something diff
  • when mutated in humans manifests in non cycling cells
53
Q

What did yeast-two hybrid show TDP1 interacts w/?

A
  • lig3a
  • physically coupled to SSBR machine
  • DIAG*
54
Q

How would you demonstrate that TDP1 req for SSBR in cells?

A
  • measure SSBR in cells –> comet assay
  • treat cells, embed in agarose, run electrophoresis
  • cells w/ more DNA damage form tail (DNA migrates)
  • measure intensity in tail and extent of migration w/ computer software, this can roughly quantify no. breaks
  • if in alkaline conditions get SSBs and DSBs
  • if in neutral conditions only DSBs
55
Q

What is a comet assay also known as?

A
  • single-cell gel electrophoresis
56
Q

What is CPT used for?

A
  • track these breaks as is a TOP1 inhibitor

- used freq in cancer and in lab to traps TOPs (so get more TOPs when more CPT)

57
Q

How and why do unrepaired SSBs (DNA-TOP1/DNA-AMP) cause disease?

A
  • if RNA pol transcribing across and hits DNA-TOP1/DNA-AMP adducts, becomes stuck, stalls transcrip, so not enough transcripts, less prot, then neurons die
  • or because PARP1 marks breaks, and uses NAD, but if too many SSBs then uses too much NAD, depletes supply, energy levels decreased, cell cant cope w/ energy demanding processes (–> overactivated PARP)
  • both cause neurodegen problems, get worse w/ age
  • can also affect DNA rep, but doesn’t affect non cycling cells –> could cause neurodev problems, as lots of unrep SSBs during neurogenesis (NOT neurodegen)
58
Q

How can you examine how unrepaired SSBs lead to disease in lab, and how can you ensure the signal is specific?

A
  • if induce lots of SSBs
  • have 3 components: PARP1, NAD, pADPr
  • measure conc of NAD (should decrease) or pADP2 (should increase)
  • Abs available for these polymers
  • ensure signal specific by KO PARP1 and see if causes signal to stop
59
Q

Is role of PARP1 physiologically signif in unrepaired SSBs?

A
  • when KO XRCC1 affects cerebellum
  • want to test if this due to PARP1 hyperactivation –> if do double mutant, then should rescue neurons (so neurons dying in XRCC1 KO are dying due to PARP1)
60
Q

So would you predict that XRCC1 mutations cause human disease?

A
  • yes, as all experiments are carried out in mice, a structurally similar model
  • XRCC1 is essential in humans
61
Q

How can deficiency in XRCC1, an essential prot result in a viable human?

A
  • have v small amount, enough to keep them alive, but not enough to keep cerebellar alive –> cerebellar degen
62
Q

Why do SSBs primarily impact NS?

A
  • cycling cells have ways to deal w/ SSBs, but non-cycling cells don’t
  • NS cells can’t regen, unlike other non-cycling cells, eg. muscle cells
63
Q

Why do SSBs primarily affect cerebellum in NS?

A
  • no. neurons in this region v high (even though small area)
  • doesn’t have other repair mechanisms, so dep on SSBR (no back ups)
  • genetic pathways diff
  • epigenetic diffs, could be same genes but exp diff
  • gene positioning in nucleus diff, affects exp
64
Q

What were the results of a comet assay w/ TDP1 present/not present?

A
  • given drug so start w/ almost 100% DNA breaks in both cell line
  • remove drug and see to what extent cells can repair damage
  • both decrease over time, but TDP1 +/+ decreases to almost 0 (-/- only to around 25% after 120 mins)
  • even in absence of TDP1 cells can repair, just less efficient –> so other things exist as well as TDP1
65
Q

How can you examine redundancy and backup pathways?

A
  • functional complementation
66
Q

How is functional complementation carried out?

A
  • use yeast strain lacking pathway of interest
  • transform w/ human DNA lib
  • plate w/ DNA damage agent
  • nothing will grow if lack pathway
  • but if complement can grow, as taken DNA that allowed it to grow = resistant clones
  • break up, take DNA, seq it
67
Q

In what organisms can functional complementation be carried out in?

A
  • can do w/ any organism, but easiest in yeast
68
Q

How is TTRAP similar to TDP1?

A
  • also important in cell DNA repair, and also induced by CPT
  • similar to TDP1 –> both active in cleaving 3’ phosphodiester bond
  • but TTRAP much less active that TDP1
69
Q

How can the role of TTRAP be further checked?

A
  • knockdown of TTRAP (ie. look at cells w/o it) and comet assay following CPT treatment
  • OR look at other substrates w/ same bond but slightly diff to see if more active
70
Q

How do the positions of Top1 and 2 vary?

A
  • Top1 on 3’ terminus of DNA and Top2 on 5’ terminus

- similar bond, but a diff polarity

71
Q

What suggested that TTRAP interacted w/ Top2 and not Top1?

A
  • TTRAP more active on 5’ ter than 3’
72
Q

How can you be sure the activity is specific to enz you are incubating and NOT a contaminant during the prep (TTRAP)?

A
  • check TTRAP causing activity by making a mutant predicted to be catalytically inactive, then purify it, if activity gone then know TTRAP was causing activity
  • best method for any prot in yeast/bacteria etc
73
Q

How can you confirm 5’ TDP1 activity in cell extracts?

A
  • prep extracts from WT cells and cells that lack TTRAP
  • mix w/ TDP1 substrate
  • as incubation time increases, see conversion of substrate to product
  • but in cells w/o TTRAP can’t process substrate well
  • to measure DSBs in single cells can do:
  • -> neutral comet assay
  • -> or immunofluorescence and look at markers of DSBs
  • -> use γ-H2AX Ab
  • -> or use drugs that specifically induce the damage you’re interested in, eg. CPT inhibits TOP1, or ETOP (etoposide) inhibits TOP2
74
Q

WHat are CTP and ETOP both used for?

A
  • to treat cancer
75
Q

What are the diffs between TOP1 and TOP2?

A
  • TOP1:
  • -> bond cleaved by TDP1
  • -> inhibitors used in cancer therapy, eg. topotecan, CPT
  • -> mutations lead to cerebellar degen
  • TOP2
  • -> bond cleaved by TTRAP = TDP2
  • -> inhibitors used in cancer therapy, eg. ETOP
  • -> unsure if mutated in neurodegen disorders
76
Q

Why is TDP2 important?

A
  • predicted to be important in non-cycling cells, as 1 way to remove prot from DNA is to cut DNA, prod structures which can be ligated by NHEJ (error prone) or HR (error free)
  • -> in non cycling cells have to use NHEJ
  • but if don’t cut DNA, and instead neatly clip bond, can use NHEJ which doesn’t involve ligation, so is error free –> good option in neurons
  • so if don’t have TDP2 would expect defects in neurons
77
Q

What happens to prot in mutation in TDP2?

A
  • prod prot lacking active site, therefore inactive
78
Q

How can you detect TDP2 activity in patient samples?

A
  • use TOP2 substrates w/ pY at 5’ termini
  • also TOP1 substate w/ pY at 3’ termini to detect TDP1
  • mix w/ extracts from patient lymphoblast cells
  • see band shift w/ substrate
  • confirm this by running extract to do WB
79
Q

How can you look for stalled transcrip (if breaks blocking transcrip), ie. measuring transcription competency?

A
  • label RNA w/ something can see
  • dep on click chemistry
  • use ribonucleotide coupled to fluorophore so can see in cells
  • incubate cells w/ EU (mod of uracil)
  • do click chem to coupled EU w/ fluorophore
  • so any uracil incorp into RNA will show signal
  • if cells cannot recover after ETOP treatment, then likely lacking TDP2
  • WT cells are able to recover
80
Q

How can you study DNA repair at the whole organismal level?

A
  • mouse → has good conservation of tissues from humans
81
Q

Which component of pathway would you choose to delete in mice?

A
  • something not essential, eg. involved in repair of only certain components –> Tdp2 KO mice
  • issues if do something essential as may not have viable mouse → but can learn more
82
Q

What is the result of a TDP2 KO mice?

A
  • > 50 genes dereg are assoc w/ neurological disease in humans
  • shows this KO affects lots of genes
83
Q

How can XRCC1 or Lig3α KO mice (essential) be made?

A
  • conditional KO: Cre Lox system
  • flank gene w/ 2 LoxP sites
  • cross w/ another mouse exp Cre recombinase
  • Cre will delete anything between 2 LoxP sites
  • so gene only deleted when Cre exp, control from promoter of Cre, so can use tissue or time specific promoter to control
  • tissue specific: most common promoter = nestin, only exp in NS, so Cre only exp if nestin exp (ie. in NS), so only delete essential gene in NS, CAN be compatible w/ embryonic viability (but not always)
  • time specific (temporal control): eg. doxycycline promoter, Cre only expressed when feed the mice w/ something that can activate promoter, so gene expressed t/o embryogenesis, then activate at some time after birth, then deleted everywhere, but viable mouse as bypassed its deletion in embryonic dev
84
Q

What does DNA Lig3α deficiency result in, and what were the consequences of this?

A
  • results in pronounced cerebellar defects
  • so could delete only in brain to study
  • mutated mice show signs of degen and motor problems
  • lig3α acts as a glue and only ligase in mt → mtDNA repair is v important too
85
Q

What are the main diffs between nDNA and mtDNA?

A
  • mt contain 2-10 copies of mtDNA and each euk cell has approx 100 mt, but can be up to 1000 –> dep on energy status of tissue (eg. muscle has more)
  • mtDNA v prone to DNA damage
86
Q

Why is mtDNA v prone to DNA damage?

A
  • mt prod most of cells ROS and mtDNA located v close to sites of ROS prod
  • lacks protective histones
  • almost exclusively transcribed (v few non coding regions)
87
Q

Why is is important to maintain mtDNA genome integrity?

A
  • controls apoptosis
  • energy prod → prod 65kg ATP/day
  • also leads to ROS prod
88
Q

How is mtDNA genome instability assoc w/ ageing?

A
  • as age, mt function decreases and more ROS species leak out of mt
89
Q

What are the consequences of unrepaired DNA damage, and how can this be avoided?

A
  • cell death
  • cell survival –> cancer
  • but if inhibit DNA repair, can induce cell death and stop cancer from dev
90
Q

What could personalised therapy pot do if target TDP1/2?

A
  • inhibit TDP1/2
  • if given at lower doses in LT can cause progressive neurological disorders, but if given acutely in ST can treat cancer
91
Q

What is synthetic lethality?

A
  • combo of deficiencies in exp of 2 or more genes leads to cell death, but a deficiency in only 1 does not
92
Q

Can defective prots of mutated DDR genes be directly targeted?

A
  • apart from attempts to stab p53 mutant, unlikely to have therapeutic benefit
93
Q

How can defective prots of mutated DDR genes be targeted indirectly?

A
  • therapeutic interventions must be targeted towards other prots w/ functions mostly dispensable in normal cells, but become essential (or at least, important) in context of DDR mutation –> therefore providing SSL
94
Q

How do mutations in cancer types differ?

A
  • each type has its own signature mutations
95
Q

What happens to a SSB at rep?

A
  • when fork hits SSB, collapses and causes DSB (if not repaired)
  • then need HR for repair –> repairs DSBs during rep
96
Q

What components does HR req?

A
  • Rad51 is a marker and BRCA2 is req
97
Q

What is a strategy to treat cancer by exploiting the concept of synthetic lethality?

A
  • inhibiting SSBR in cancers deficient in HR, eg. by inhibiting PARP in BRCA1 deficient cancers (BRCA2-/- sensitive to PARP inhibitors, but BRCA2+/- are insensitive)
  • so if inhibit PARP and this was all they needed then cells would die, test this through clonogenic survival assay
  • test strategy experimentally by:
  • -> inhibiting SSBR, should channel SSBs to HR, this overactivation of HR can be measured by HR markers (eg. Rad51)
  • -> or knockdown PARP1 in HR deficient cells, which increases HR
98
Q

How do clonogenic survival assays work, and when are they freq used?

A
  • more colonies count the more the treatment leads to survival
  • used freq for cancer drugs
99
Q

Are PARP KO mice normal?

A
  • healthy –> if delete 1 PARP others can compensate, in cancer need non essential target, otherwise v toxic, so PARP is good target
100
Q

What drug has now been approved to target PARP?

A
  • Olaparib
  • highly specific
  • no need for other DNA damaging agents
101
Q

How do cancer cells adapt and resist treatment over time?

A
  • switching off target (eg. w/ TOP poisons, PARP inhibitors)
  • upreg 1° repair mech
  • upreg parallel repair mech –> eg. HR is main pathway to repair DSBs, but NHEJ is an alt pathway that can be activated, so not dep on HR
  • epigenetic activation –> change histone marks that reg exp of certain prots
102
Q

How can we overcome this adaptation of cancer cells to resist treatment?

A
  • understand more about 1° and parallel “redundant” repair mechs
103
Q

How is DNA damage connected to autoinflam and innate immunity?

A
  • DNA is compartmentalised w/in nucleus or in mt, to prevent autoimmunity
  • but despite this, cGAS activated by DNA damage
104
Q

What is the role of cGAS, and how is it connected to inflam?

A
  • cytosolic sensor of dsDNA
  • if dsDNA in cyto, senses it and makes cGAMP
  • cGAMP important ligand in activating STING (stimulator of IFN genes) pathway
  • so if have dsDNA in cyto leads to activation of c-GAS STING pathway, which activates IFN genes, which are mediators of inflam
105
Q

What products of DNA damage can cGAS also fight?

A
  • eg. ribonucleotides incorp into DNA

- RNase H2 needed for incision of ribonucleotides

106
Q

What does having no RNase H2 cause?

A
  • causes neuroinflammatory disorder
  • eg. Aicardi-Goutieres Syndrome –> senses DNA as if foreign, so must have escaped nucleus/mt, seems to mimic congenital viral infection
107
Q

How does a defect in a DNA repair enz cause autoinflam?

A
  • when parental cell divides, if lots of unrepaired DNA damage causes lagging strand to lag behind a bit, so these chroms lag behind and compartmentalised into micronuclei upon division
  • if have RNase H2 have lower levels of micronuclei
  • but micronuclei have their own membrane, so cyto sensor sees DNA inside them, as micronuclei attracted to cGAS
108
Q

What is the consequence of that fact that micronuclear DNA is particularly susceptible to DNA damage?

A
  • leads to chromothripsis
  • occurs as a consequence of irreversible nuclear envelope collapse, which arises freq in micronuclei due to defective nuclear lamina organisation
109
Q

What experiment was carried out to test if cGAS assoc w/ micronuclei upon increased DNA damage?

A
  • see DNA damage in cells using γ-H2AX as a marker of DNA breaks, so see if colocalisation between marker and cGAS
  • count % of γ-H2AX +ve micronuclei
  • cGAS +ve micronuclei have more γ-H2AX –> so cGAS localisation to micronuclei linked to DNA damage
110
Q

What experiment was carried out to test if cGAS-STING signalling pathway is activated upon DNA damage?

A
  • take some cells and treat w/ DNA damage agent (eg. CTP, ionising radiation)
  • segregate cells which are micronuclei +ve and -ve
  • see if activation of pathway by genome wide analysis –> eg. RNA-seq, RNA FISH
  • those w/o micronuclei won’t activate cGAS as well as those w/
  • micronuclei +ve have ISG upreg
  • micronuclei -ve have low ISG
111
Q

What is the diff between using RNA-seq and RNA FISH?

A
  • RNA-seq can sep micronuclei +ve and -ve cells, as have diff exp of eg. IFN genes
  • single cell RNA FISH –> not genome wide, design specific probes
112
Q

What do micronuclei provide a source of?

A
  • substantial source of immunostimulatory DNA
113
Q

What are the implications of micronuclei in neurological disorders and cancer?

A
  • neurological disorders –> leakage of n/mtDNA can activate cGAS-STING causing autoinflam in NS
  • cancer –> micronuclei freq form in cancer cells, cGAS may often be activated by this route during neoplastic formation, leading to cGAS-dep and STING-dep tumour suppressive immune responses
114
Q

How can role of micronuclei be used to treat cancer?

A
  • may be selection pressures during cancer evo to inactivate cGAS-STING signalling
  • it’s freq inactivated in tumours
  • could this be used in immunotherapy?
115
Q

How can PARP1 inhibitors be used to treat cancer?

A
  • used to treat RNaseH2 deficient cancers

MBB313 - Genome Stability and Genetic Change (3 decks)

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