Brainscape's Knowledge GenomeTM

Browse over 1 million classes created by top students, professors, publishers, and experts.


See full index

genome stability and genetic change > Khamisy > Flashcards

Khamisy Flashcards

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
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
2
Q

why can DNA breaks be good

A

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

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
3
Q

what are the consequences of unrepaired DNA damage

A

cell death

cell survival - lead to mutations and cancer

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
4
Q

what do DNA topoisomerases do?

A

make an intentional break in DNA to untangle it

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
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

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
6
Q

how can you visualise R loops in the lab

A

using s9.6 antibody in immunostaining

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
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)

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
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

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
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)

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
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

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
11
Q

what enzyme acts to cleave ribonucleotides from RNA:DNA complexes

A

RNaseH2

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
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

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
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

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
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

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
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

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
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

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
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

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
18
Q

how can DNA breaks be a friend

A

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

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
19
Q

what is the main consequence of base deamination

A

mispairing - CG-AT transition

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
20
Q

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

A

single strand breaks

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
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)

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
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

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
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

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
24
Q

what is the final step to single strand DNA break repair

A

ligation - DNA ligase seals the nicks

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
25
Q

what protein would be good to study the process of SSBR

A

XRCC1 as it is seen at many steps and is a scaffold

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
26
Q

what are the principles behind yeast-2-hybrid screens to study protein protein interactions

A

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 well did you know this?
1
Not at all
2
3
4
5
Perfectly
27
Q

how can you confirm the results of a yeast-2-hybrid

A

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

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
28
Q

when you find that a protein interacts with your protein of interest what should you do next

A

ask what this protein is
do a blast search and get an idea about the domain structure of the protein
do a pubmed search

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
29
Q

what do mutation in aprataxin cause

A

ataxia oculomotor apraxia-1 (AOA1)

  • variable mental retardation
  • cerebellar degeneration
  • spinocerebellar ataxia
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
30
Q

how do you study the function of a protein in the lab

A

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

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
31
Q

aprataxin contains a HIT site that has ADP linking it to DNA. when labelled DNA with AMP attached was incubated with aprataxin what happens?

A

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 well did you know this?
1
Not at all
2
3
4
5
Perfectly
32
Q

how do DNA-AMP adducts arise in cells

A

through premature abortion of a ligase reaction. Final step of ligation doesnt take place when there is loss of the 3’ OH group

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
33
Q

what does aprataxin do

A

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

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
34
Q

what is an example of an endogenous threat that can lead to abortive ligation reactions and therefore DNA-AMP adducts

A

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

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
35
Q

what are the major steps to SSBR

A
  • recongition (PARP)
  • DNA end processing
  • gap filling with polymerase
  • sealing with ligase
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
36
Q

what are the steps to be taken following a genetic screen

A

blast search and look at pubmed to see what is known about the domains

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
37
Q

how can you ascertain that aprataxin processes DNA-AMP adducts in vivo

A

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
Q

what do mutation in TDP1 encoding topoisomerase-I dependent DNA damage repair enzyme cause

A

spinocerebellar ataxia with axonal neuropathy (SCAN1)

39
Q

what are topoisomerases

A

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
Q

what happens in abortive topoisomerase events

A

topoisomerase protein remains crosslinked to the DNA (DNA-protein crosslinks)

41
Q

what causes abortive topoisomerase events

A

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
Q

what protein does TDP1 interact with

A

Lig3a

43
Q

what is comet assay

A

(single cell gel electrophoresis) is a method used to measure DNA strand breaks in DNA

44
Q

what does alkaline comet allow you to measure

A

both SSB and DSB as it denatures the two strand

45
Q

what does neutral comet assay allow you to measure

A

only DSB

46
Q

how and why do unrepaired SSB (DNA-TOP1 or DNA-AMP) cause disease

A
  • 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
Q

what does TDP1 do

A

relieves abortive topoisomerase events

48
Q

how is TDP1 physically coupled to the SSBR machinery

A

via Lig3

49
Q

XRCC1 deficiency can also cause human disease. How can you have a viable human with a deficiency in an essential protein such as XRCC1

A

Sufficient expression to allow embryonic development and viability but not sufficient to maintain proper neurological function during life time

50
Q

why do SSB primarily affect the nervous system

A

neurons are non replicating - no mechanism of dealing with the breaks

51
Q

why do SSB primarily impact the cerebellum

A

cells without TDP1 can still repair albeit with less efficiency. Cerebellum may not have these redundant pathways and may be reliant on TDP1

52
Q

what is functional complementation

A

allows you to discover new gene functions that can complement in the absence of other gene functions

53
Q

how do you carry out a functional complementation experiemtn

A

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
Q

what was shown to be able to functional complement TDP1

A

TTRAP

55
Q

what is the difference between type I and type II topoisomerases

A

type I - attaches to 3’ terminus of transient SSB

type II - attaches to 5’ terminus of transient DSB

56
Q

how can you be sure the biochemical activity is specific to the enzyme you’re incubating and not a contaminant during preparation

A

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
Q

what was TTRAP renamed to

A

TDP2

58
Q

what is the difference between TDP1 and TDP2

A

the polarity - TDP1 prefers the 3’ terminus from top1

TDP2 prefers 5’ terminus from Top2

59
Q

what does mutation in exon 3 of TDP2 cause

A

intron retention, exon skipping, alternative splcing. Leads to truncation of TDP2 and loss of active site

60
Q

what happens if TDP2 is unable to function

A

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
Q

how can you detect TDP2 activity

A

incubate cell extract with substrate that is linked to topo and look for band shift

62
Q

how would you determine whether absence of TDP2 compromises the transcription ability of cells

A

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
Q

what are 2 examples of approaches to measure DNA-double strand breaks

A

neutral comet assay

gammaH2AX immunostaining

64
Q

TDP2 provides an error free pathway for repairing DSB

A

T. Without it nucleases are required to repair which causes loss of genetic material

65
Q

what is a conditional knockout

A

technique used to eliminate a gene in certain tissues

66
Q

how are conditional knockouts achieved

A

using the cre-lox system

67
Q

explain the cre lox system

A

the gene of interest is flanked with 2 LoxP sites

transgenic mice can be generated that induce the expression of cre recombinase

68
Q

what does Lig3a deficiency result in

A

pronounced cerebellar defects

69
Q

lig3a is the only ligase in what organelle

A

mitochondria. (no back up pathway)

70
Q

what are the main differences between nuclear DNA and mtDNA

A 
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
Q

what do chemo/radio therapy rely

A

inhibiting DNA repair on inducing DNA damage

72
Q

what is synthetic lethality

A

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
Q

what does synthetic lethality commonly target in cancer cases

A

proteins that have functions that are dispensible in normal cells but become essential in the DDR mutations of cancer cells

74
Q

defects in which pathway are common in breast cancer

A

homologous recombination

75
Q

what happens to an unrepaired SSB at replication

A

is converted to a DSB and has to be repaired by HR

76
Q

what is a synthetic lethality prinicple to treat cancer cells that are defective in HR

A

inhibit SSBR by inhibiting eg PARP in BRCA1 deficient cancers that are unable to carry out HR

77
Q

how can cancer cells adapt to synthetic lethality treatment

A
  • switch off the target (eg downregulation of PARP)
  • upregulating the primary repair mechanism
  • upregulating a parallel repair mechanism
  • epigenetic adaptation
78
Q

what is the cytosolic sensor of dsDNA

A

cGAS - leads to activation of the sting pathway to mediate inflammation

79
Q

why does DNA have to be compartmentalised

A

DNA is a key pathogen-associated molecular pattern that is sensed by the innate immune system

80
Q

cGAS activation by the presence of cytosolic DNA damage generates what

A

cGAMP which in turn induces a type I interferon response via the adaptor sting

81
Q

cGAS sting-dependent inflammation is associated with mutations in what

A

mutliple nucleases

82
Q

when RNase H2 is mutated and defective what can be caused

A

Aicardi-Goutieres syndrome - has puffy red lesions caused by inflammation
- also have the appearance of more micronuclei

83
Q

micronuclei are particularly susceptibly to what

A

DNA damage

84
Q

how would you design an experiment to test if cGAS associates with micronuclei upon increased DNA damage

A

label with fluorescence for cGAS and H2AX (dna damage)

see if these signals colocalise in cells that are deficient in RNase H2

85
Q

Design an experiment to test if the cGAS-STING pathway is activated upon DNA damage

A

expose cell to ionising radiation

  • RNA-seq of interferon response genes
  • subject micronuclei positive and negative to RNA-seq and compare the expression
86
Q

cells with micronuclei have more expression of the interferon response genes T/F

A

T

87
Q

what is another way of measuring the expression of genes apart from RNA-seq

A

single cell RNA FISH

- design hybridisation probes to specific interferon response genes

88
Q

micronuclei frequently form in _____ cells, leading to cGAS and STING dependent _____ supressive immune responses

A

cancer

tumour

89
Q

there may have been a selection pressure during cancer evolution to inactivate the cGAS-STING pathway. it is frequently inactivated in tumours T/F

A

T

90
Q

what is another example of synthetic lethality

A

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
Q

how can cancer cells deficient in RNase H2 become resistant to PARP1 inhibition

A

downregulate parp1 or top1

genome stability and genetic change (1 decks)

Brainscape helps you reach your goals faster, through stronger study habits.
© 2024 Bold Learning Solutions. Terms and Conditions