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genome stability and genetic change > Khamisy > Flashcards

Khamisy Flashcards

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1
Q

DNA breaks can come in different types. mention some

A 
single stranded DNA breaks
Base damage
bulky dna lesions
crosslinks
double strand breaks
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2
Q

why can DNA breaks be good

A

generate immunoglobin diversity
in meiosis - generates diversity
Control the transcription of genes

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3
Q

what are the consequences of unrepaired DNA damage

A

cell death

cell survival - lead to mutations and cancer

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4
Q

what do DNA topoisomerases do?

A

make an intentional break in DNA to untangle it

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5
Q

how can the process of transcription be a source of DNA damage

A

RNA pol II generates positive supercoils in front of the polymerase and negative behind it. RNA can pair with the relaxed DNA in the negative supercoils and form R loops - a major source of genomic instability

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6
Q

how can you visualise R loops in the lab

A

using s9.6 antibody in immunostaining

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7
Q

how can transcriptional R loops arise

A

from physiological and pathological sources:
repetitive regions and G rich sequences favour R loop formation.
rDNA sequences, common fragile sites
pathological repeat expansions (C9orf72 ALS, spinocerebellar ataxias, friedrich ataxia)

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8
Q

what are the consequences of R loops

A

if they form at the end of a DNA strand they can be useful to guide termination of transcription
can relieve topological constrains and supercoiling
major source of DNA breaks if left unresolved

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9
Q

why is the presence of ribose in DNA very dangerous

A

presence of a single ribose embedded in DNA makes the phosphate bond very labile. Oxygen from the ribose can attack the phosphodiester bond and cleave it (nucleophilic attack)

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10
Q

how/why does ribose contamination take place

A

erroneous activity of DNA polymerases. The relative abundance of ribonucleotide triphosphates is at least 2 orders of magnitude greater than abundance of deoxyribonucleotide triphosphates. Chance that by mistake that the DNA polymerase incorporates e-NTPs during DNA synthesis

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11
Q

what enzyme acts to cleave ribonucleotides from RNA:DNA complexes

A

RNaseH2

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12
Q

what can spontaneous base loss lead to

A

abasic sites that can block DNA replication and transcription. These abasic sites are very labile under physiological conditions

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13
Q

give an example of an endogenous base modification

A

cytosine can lose its amino group by spontaneous deamination to form uracil. This can cause mispairing

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14
Q

what happens if modified bases such as a cytosine becoming uracil are not repaired

A

During DNA replication can see a transition from GC pairing from the original DNA before modification to AT

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15
Q

give 2 examples of another endogenous deamination reaction that results in modified bases

A

guanine converts to Xanthine which can’t base pair

5’methylcytosine can convert to thymine which leads to a GC-AT transition

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16
Q

what are some of the consequences of oxidation

A

can cause thymine to form thyine glycol

oxidation of guanine causes formation of 8-oxoguanine which can lead to GC to TA mutation

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17
Q

DNA is under contant threat endogneously and exogenously. Name some of these threats

A

End - replication, transcription, ribose, contamination, reaction with molecules in the cell such as water and oxygen
exo - reaction with molecules outside the cell, cosmic rays, man-made chemicals

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18
Q

how can DNA breaks be a friend

A

can regulate gene expression and can generate diversity in immunoglobin production and/or meiosis

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19
Q

what is the main consequence of base deamination

A

mispairing - CG-AT transition

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20
Q

are single strand breaks or double strand breaks more common in cells

A

single strand breaks

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21
Q

what is the first stage of the repair of single strand breaks and what carries it out.

A

damage detection

carried out by polyADP ribose (PARP)

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22
Q

what is the second step of the repair of single stranded breaks and why is it required

A

end processing

required to restore the chemistry in the DNA broken ends

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23
Q

what is the third step of the repair of single strand DNA breaks

A

gap filling - need DNA polymerases to fill the gap

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24
Q

what is the final step to single strand DNA break repair

A

ligation - DNA ligase seals the nicks

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25
what protein would be good to study the process of SSBR
XRCC1 as it is seen at many steps and is a scaffold
26
what are the principles behind yeast-2-hybrid screens to study protein protein interactions
Fuse bait protein to Gal4 binding domain fuse prey protein to Gal4 activating domain if the protein interact there will be transcription of a reporter gene (such as b-galactosidase) under Gal4 promoter
27
how can you confirm the results of a yeast-2-hybrid
switch round which protein is on the bait and prey | do a coimmunoprecipitation, pull one protein down using an antibody and see if the other protein comes with it
28
when you find that a protein interacts with your protein of interest what should you do next
ask what this protein is do a blast search and get an idea about the domain structure of the protein do a pubmed search
29
what do mutation in aprataxin cause
ataxia oculomotor apraxia-1 (AOA1) - variable mental retardation - cerebellar degeneration - spinocerebellar ataxia
30
how do you study the function of a protein in the lab
can engineer protein into a plasmid and transformed into e.coli cells are induced to produce the protein using IPTG when cells are lysed can purify this protein away using things like a nickle column
31
aprataxin contains a HIT site that has ADP linking it to DNA. when labelled DNA with AMP attached was incubated with aprataxin what happens?
see a band shift of AMP-DNA to DNA with increasing concentrations of aprataxin. this shows that aprataxin has a role in cleaving AMP from AMP-DNA
32
how do DNA-AMP adducts arise in cells
through premature abortion of a ligase reaction. Final step of ligation doesnt take place when there is loss of the 3' OH group
33
what does aprataxin do
cleaves AMP from DNA following abortive ligase reactions to reset the cell cycle and give the cell time to repair the 3'OH bond so ligation can fully take place
34
what is an example of an endogenous threat that can lead to abortive ligation reactions and therefore DNA-AMP adducts
formation of ribonucleotide (DNA-RNA). RNaseH2 incises this and it is repaired by RER if DNA ligase prematurely attempts to ligate will have the formation of a DNA-AMP adduct - aprataxin gives another chance for RER
35
what are the major steps to SSBR
- recongition (PARP) - DNA end processing - gap filling with polymerase - sealing with ligase
36
what are the steps to be taken following a genetic screen
blast search and look at pubmed to see what is known about the domains
37
how can you ascertain that aprataxin processes DNA-AMP adducts in vivo
2 cells - WT and aprataxin knoxkout extract DNA from both cells and incubate with recombinant aprataxin centrifuge - expect more AMP to be present in the supernatant of AMP knockout bc it originally had more AMP bound to DNA measure how much AMP using mass spec
38
what do mutation in TDP1 encoding topoisomerase-I dependent DNA damage repair enzyme cause
spinocerebellar ataxia with axonal neuropathy (SCAN1)
39
what are topoisomerases
enzymes that relax superhelical tension that arise from the compaction of DNA into very tiny space. Makes a break in strand of DNA to allow the other strand to swivel around a reseal
40
what happens in abortive topoisomerase events
topoisomerase protein remains crosslinked to the DNA (DNA-protein crosslinks)
41
what causes abortive topoisomerase events
misalignment of the 3'OH - can take place due to certain drugs, nearby presence of other oxidative breaks or collision of transcription machinery with the N terminus
42
what protein does TDP1 interact with
Lig3a
43
what is comet assay
(single cell gel electrophoresis) is a method used to measure DNA strand breaks in DNA
44
what does alkaline comet allow you to measure
both SSB and DSB as it denatures the two strand
45
what does neutral comet assay allow you to measure
only DSB
46
how and why do unrepaired SSB (DNA-TOP1 or DNA-AMP) cause disease
- RNA pol may hit SSB and result in stalled transcription - excessive PARP activation (uses NAD as a substrate - leading to energy deficiency in cell) - can both resut in neurodegenerative disorders - can both result in collapsed DNA replication
47
what does TDP1 do
relieves abortive topoisomerase events
48
how is TDP1 physically coupled to the SSBR machinery
via Lig3
49
XRCC1 deficiency can also cause human disease. How can you have a viable human with a deficiency in an essential protein such as XRCC1
Sufficient expression to allow embryonic development and viability but not sufficient to maintain proper neurological function during life time
50
why do SSB primarily affect the nervous system
neurons are non replicating - no mechanism of dealing with the breaks
51
why do SSB primarily impact the cerebellum
cells without TDP1 can still repair albeit with less efficiency. Cerebellum may not have these redundant pathways and may be reliant on TDP1
52
what is functional complementation
allows you to discover new gene functions that can complement in the absence of other gene functions
53
how do you carry out a functional complementation experiemtn
take yeast strain which is mutated in pathway you're interested in. Have a human cDNA library that represents most of human coded genes. Transfect library and plate on media that is exposed to a DNA damage agent (CPT) take resistant clones, smash them open and sequence the plasmids to discover the new gene that complements
54
what was shown to be able to functional complement TDP1
TTRAP
55
what is the difference between type I and type II topoisomerases
type I - attaches to 3' terminus of transient SSB | type II - attaches to 5' terminus of transient DSB
56
how can you be sure the biochemical activity is specific to the enzyme you're incubating and not a contaminant during preparation
the enzyme purified in a lab may still have a contaminant. make it catalytically inactive by mutating the active site and purify - if you still get the activity then it is probs the contaminant thats causing the activity. If not, you can see it was the enzyme
57
what was TTRAP renamed to
TDP2
58
what is the difference between TDP1 and TDP2
the polarity - TDP1 prefers the 3' terminus from top1 | TDP2 prefers 5' terminus from Top2
59
what does mutation in exon 3 of TDP2 cause
intron retention, exon skipping, alternative splcing. Leads to truncation of TDP2 and loss of active site
60
what happens if TDP2 is unable to function
without TDP2, nucleases act to remove the links with topo. This can result in the loss of genomic info.The break is then repaired by error prone NHEJ as noncycling cells don't have sister chromatids for HR
61
how can you detect TDP2 activity
incubate cell extract with substrate that is linked to topo and look for band shift
62
how would you determine whether absence of TDP2 compromises the transcription ability of cells
label newly formed RNA with a metabolic label eg by incubating proliferating cells with 5-EU. This analogue can be coupled with a fluorophore using click chemistry
63
what are 2 examples of approaches to measure DNA-double strand breaks
neutral comet assay | gammaH2AX immunostaining
64
TDP2 provides an error free pathway for repairing DSB
T. Without it nucleases are required to repair which causes loss of genetic material
65
what is a conditional knockout
technique used to eliminate a gene in certain tissues
66
how are conditional knockouts achieved
using the cre-lox system
67
explain the cre lox system
the gene of interest is flanked with 2 LoxP sites | transgenic mice can be generated that induce the expression of cre recombinase
68
what does Lig3a deficiency result in
pronounced cerebellar defects
69
lig3a is the only ligase in what organelle
mitochondria. (no back up pathway)
70
what are the main differences between nuclear DNA and mtDNA
``` mito DNA is prone to damage - mitochondria produce most of the cells ROS - lacks protective histones - no non-coding regions each cell has multiple copies of mtDNA ```
71
what do chemo/radio therapy rely
inhibiting DNA repair on inducing DNA damage
72
what is synthetic lethality
the exploitation of genetic defects essential for tumour cell survival by combining defect in an affected pathway with a pharmacologically induced defect in a compensatory way
73
what does synthetic lethality commonly target in cancer cases
proteins that have functions that are dispensible in normal cells but become essential in the DDR mutations of cancer cells
74
defects in which pathway are common in breast cancer
homologous recombination
75
what happens to an unrepaired SSB at replication
is converted to a DSB and has to be repaired by HR
76
what is a synthetic lethality prinicple to treat cancer cells that are defective in HR
inhibit SSBR by inhibiting eg PARP in BRCA1 deficient cancers that are unable to carry out HR
77
how can cancer cells adapt to synthetic lethality treatment
- switch off the target (eg downregulation of PARP) - upregulating the primary repair mechanism - upregulating a parallel repair mechanism - epigenetic adaptation
78
what is the cytosolic sensor of dsDNA
cGAS - leads to activation of the sting pathway to mediate inflammation
79
why does DNA have to be compartmentalised
DNA is a key pathogen-associated molecular pattern that is sensed by the innate immune system
80
cGAS activation by the presence of cytosolic DNA damage generates what
cGAMP which in turn induces a type I interferon response via the adaptor sting
81
cGAS sting-dependent inflammation is associated with mutations in what
mutliple nucleases
82
when RNase H2 is mutated and defective what can be caused
Aicardi-Goutieres syndrome - has puffy red lesions caused by inflammation - also have the appearance of more micronuclei
83
micronuclei are particularly susceptibly to what
DNA damage
84
how would you design an experiment to test if cGAS associates with micronuclei upon increased DNA damage
label with fluorescence for cGAS and H2AX (dna damage) | see if these signals colocalise in cells that are deficient in RNase H2
85
Design an experiment to test if the cGAS-STING pathway is activated upon DNA damage
expose cell to ionising radiation - RNA-seq of interferon response genes - subject micronuclei positive and negative to RNA-seq and compare the expression
86
cells with micronuclei have more expression of the interferon response genes T/F
T
87
what is another way of measuring the expression of genes apart from RNA-seq
single cell RNA FISH | - design hybridisation probes to specific interferon response genes
88
micronuclei frequently form in _____ cells, leading to cGAS and STING dependent _____ supressive immune responses
cancer | tumour
89
there may have been a selection pressure during cancer evolution to inactivate the cGAS-STING pathway. it is frequently inactivated in tumours T/F
T
90
what is another example of synthetic lethality
in cancers that are genetically defective in RNase H2, you can inhibit PARP1 (is ribonucleotides arent repaired by RNase H2, can be sensed and cause trapping of top1 leading to single strand breaks that are substrates for PARP1)
91
how can cancer cells deficient in RNase H2 become resistant to PARP1 inhibition
downregulate parp1 or top1

genome stability and genetic change (1 decks)

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