Khamisy Flashcards
DNA breaks can come in different types. mention some
single stranded DNA breaks Base damage bulky dna lesions crosslinks double strand breaks
why can DNA breaks be good
generate immunoglobin diversity
in meiosis - generates diversity
Control the transcription of genes
what are the consequences of unrepaired DNA damage
cell death
cell survival - lead to mutations and cancer
what do DNA topoisomerases do?
make an intentional break in DNA to untangle it
how can the process of transcription be a source of DNA damage
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
how can you visualise R loops in the lab
using s9.6 antibody in immunostaining
how can transcriptional R loops arise
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)
what are the consequences of R loops
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
why is the presence of ribose in DNA very dangerous
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)
how/why does ribose contamination take place
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
what enzyme acts to cleave ribonucleotides from RNA:DNA complexes
RNaseH2
what can spontaneous base loss lead to
abasic sites that can block DNA replication and transcription. These abasic sites are very labile under physiological conditions
give an example of an endogenous base modification
cytosine can lose its amino group by spontaneous deamination to form uracil. This can cause mispairing
what happens if modified bases such as a cytosine becoming uracil are not repaired
During DNA replication can see a transition from GC pairing from the original DNA before modification to AT
give 2 examples of another endogenous deamination reaction that results in modified bases
guanine converts to Xanthine which can’t base pair
5’methylcytosine can convert to thymine which leads to a GC-AT transition
what are some of the consequences of oxidation
can cause thymine to form thyine glycol
oxidation of guanine causes formation of 8-oxoguanine which can lead to GC to TA mutation
DNA is under contant threat endogneously and exogenously. Name some of these threats
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
how can DNA breaks be a friend
can regulate gene expression and can generate diversity in immunoglobin production and/or meiosis
what is the main consequence of base deamination
mispairing - CG-AT transition
are single strand breaks or double strand breaks more common in cells
single strand breaks
what is the first stage of the repair of single strand breaks and what carries it out.
damage detection
carried out by polyADP ribose (PARP)
what is the second step of the repair of single stranded breaks and why is it required
end processing
required to restore the chemistry in the DNA broken ends
what is the third step of the repair of single strand DNA breaks
gap filling - need DNA polymerases to fill the gap
what is the final step to single strand DNA break repair
ligation - DNA ligase seals the nicks
what protein would be good to study the process of SSBR
XRCC1 as it is seen at many steps and is a scaffold
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
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
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
what do mutation in aprataxin cause
ataxia oculomotor apraxia-1 (AOA1)
- variable mental retardation
- cerebellar degeneration
- spinocerebellar ataxia
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
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
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
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
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
what are the major steps to SSBR
- recongition (PARP)
- DNA end processing
- gap filling with polymerase
- sealing with ligase
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
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
what do mutation in TDP1 encoding topoisomerase-I dependent DNA damage repair enzyme cause
spinocerebellar ataxia with axonal neuropathy (SCAN1)
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
what happens in abortive topoisomerase events
topoisomerase protein remains crosslinked to the DNA (DNA-protein crosslinks)
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
what protein does TDP1 interact with
Lig3a
what is comet assay
(single cell gel electrophoresis) is a method used to measure DNA strand breaks in DNA
what does alkaline comet allow you to measure
both SSB and DSB as it denatures the two strand
what does neutral comet assay allow you to measure
only DSB
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
what does TDP1 do
relieves abortive topoisomerase events
how is TDP1 physically coupled to the SSBR machinery
via Lig3
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
why do SSB primarily affect the nervous system
neurons are non replicating - no mechanism of dealing with the breaks
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
what is functional complementation
allows you to discover new gene functions that can complement in the absence of other gene functions
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
what was shown to be able to functional complement TDP1
TTRAP
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
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
what was TTRAP renamed to
TDP2
what is the difference between TDP1 and TDP2
the polarity - TDP1 prefers the 3’ terminus from top1
TDP2 prefers 5’ terminus from Top2
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
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
how can you detect TDP2 activity
incubate cell extract with substrate that is linked to topo and look for band shift
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
what are 2 examples of approaches to measure DNA-double strand breaks
neutral comet assay
gammaH2AX immunostaining
TDP2 provides an error free pathway for repairing DSB
T. Without it nucleases are required to repair which causes loss of genetic material
what is a conditional knockout
technique used to eliminate a gene in certain tissues
how are conditional knockouts achieved
using the cre-lox system
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
what does Lig3a deficiency result in
pronounced cerebellar defects
lig3a is the only ligase in what organelle
mitochondria. (no back up pathway)
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
what do chemo/radio therapy rely
inhibiting DNA repair on inducing DNA damage
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
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
defects in which pathway are common in breast cancer
homologous recombination
what happens to an unrepaired SSB at replication
is converted to a DSB and has to be repaired by HR
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
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
what is the cytosolic sensor of dsDNA
cGAS - leads to activation of the sting pathway to mediate inflammation
why does DNA have to be compartmentalised
DNA is a key pathogen-associated molecular pattern that is sensed by the innate immune system
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
cGAS sting-dependent inflammation is associated with mutations in what
mutliple nucleases
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
micronuclei are particularly susceptibly to what
DNA damage
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
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
cells with micronuclei have more expression of the interferon response genes T/F
T
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
micronuclei frequently form in _____ cells, leading to cGAS and STING dependent _____ supressive immune responses
cancer
tumour
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
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)
how can cancer cells deficient in RNase H2 become resistant to PARP1 inhibition
downregulate parp1 or top1
