Quiz 2 (Lec 6-7) Flashcards
1
Q
is DNA very stable?
A
- wrong assumption
- DNA is susceptible to damage: nicks, breaks, base alterations, chemical reactions
- repair mechanisms prevent damage from becoming mutations
2
Q
endogenous vs exogenous DNA damage
A
- endogenous = spontaneous
- exogenous = environmental
3
Q
DNA damage vs mutation
A
- damage = physical alteration to structure of DNA
- mutation = change in sequence after replication = inherited by daughter cell, cannot be repaired
4
Q
types of DNA damage
A
endogenous:
1) depurination / depyrimidination
2) deamination
3) oxidative damage
exogenous:
1) ionizing radiation
2) UV radiation
3) alkylation
5
Q
depurination / depyrimidination
A
- loss of nitrogenous base through hydrolysis of N-glycosidic bond
- results in apurinic (more common) / apyrimidinic / abasic (AP) site = loss of coding info for DNA replication or transcription
- also risk of DSB
6
Q
DSB from depurination / depyrimidination
A
1) free OH = potential for linearization of sugar
2) beta-elimination: base attack leads to loss of 3’ connection to phosphate
3) no 3’OH to help with repair = DSB
7
Q
translesion (bypass) synthesis polymerase
A
1) replication or transcription stalls at site of damage (ex. AP site)
2) PCNA ubiquinated
3) recruitment and switch to bypass polymerase (eta), fills gap with random nucleotide
4) PCNA deubiquinated, switch back to replisome to continue process
8
Q
deamination
A
- exocyclic amine group replaced with C=O
- can occur on A, G, C
- loss of two H-bond donors, replaced with two H-bond acceptors
- ex. C –> U changes base pairing (G-C to A-U)
9
Q
oxidative damage
A
- from ROS generated in cellular processes: ETC, drug metabolism, inflammation
10
Q
ROS reactions
A
O2 + electron = O2- (superoxide)
O2- + e, 2H+ = H2O2 (hydrogen peroxide)
H2O2 + e, H+ = H2O + OH radical
OH radical + e, H+ = H2O
11
Q
protective mechanisms against ROS
A
- enzymes and anti-oxidants protect DNA, protein and lipids from ROS
- ex. SOD, catalase
12
Q
oxidative damage example: guanine
A
- guanine + OH radical = 8-oxo-G
- steric clash introduced = syn conformation preferred
- base pairs with adenine instead of cytosine
- 2nd round of replication = mutation
13
Q
ionizing radiation
A
- produces ROS (splitting water)
- also directly damages DNA bonds
14
Q
UV radiation
A
- produces pyrimidine dimers
- pi bond electrons excited by UV light, can cause stacked pyrimidines to bond
15
Q
pyrimidine stacking effects
A
1) pulls bases closer together (3.4 to 1.5A)
2) distortion (kink/bend) because of disrupted H-bonding
3) interferes with polymerase activity (binding)
16
Q
types of pyrimidine dimers
A
1) two bonds = cyclobutane pyrimidine dimer (CPD)
2) one bond = 6-4 photoproduct
17
Q
TLS in pyrimidine stacking
A
- fills gap to allow replication or transcription to continue
- preference for adding A:A dimer = potential mutation
18
Q
alkylation
A
- transfer of methyl to base
- ex. from SAM
- ex. methylation of O6 on guanine alters H-bonding, can now base pair with T
19
Q
relative frequencies of DNA damage
A
1) UV radiation: 100 000 lesions/cell/day
2) depurination: 10 000
3) alkylation: 5 000
4) depyrimidination: 500
5) deamination: 100
20
Q
how does sunscreen protect skin from DNA damage?
A
- delocalized electrons in organic compounds absorb some energy from UV radiation
- released photons are at lower energy
21
Q
DNA repair mechanisms
A
1) direct repair
2) base excision repair (BER)
3) nucleotide excision repair (NER)
4) mismatch repair
5) post-replication repair
6) DSB repair
22
Q
direct repair
A
- fixes damaged base with specific enzymes that reverse the damage
23
Q
O6-methylguanine-DNA methyltransferase (MGMT)
A
1) recognizes kink caused by G methylation
2) interdigitation: flips damaged base out into active site and inserts Arg into DNA helix to stabilize
3) Cys145 attacks methyl = suicide enzyme (degraded)
24
Q
general steps to replacing DNA damage
A
1) damaged DNA recognized by protein
2) enzymes recruited to break phosphodiester bond to remove damaged area
3) DNA polymerase rebuilds removed area
4) DNA ligase re-seals phosphodiester bonds
25
base excision repair
remove and replace damaged nitrogenous base:
1) proteins recognize changes in DNA bases
2) enzymes recruited: glycosylase breaks N-glycosidic linkage to generate an AP site (specific for each type of DNA damage)
3) endonuclease nicks backbone
4) dRP lyase removes deoxyribose
4) DNA polymerase replaces nucleotide
5) DNA ligase seals phosphodiester bond
26
nucleotide excision repair
remove and replace damaged nucleotide
1) XPC-HR23B recognizes changes in helix structure
2) TFIIH (XPB/XPD) is recruited to unwind DNA: XPC-HR23B interdigitation enhances kink severity to attract
3) RPA protects ssDNA (TFIIH bound to damaged strand only)
4) endonucleases (3' XPG/XPA and 5' XPF) cleave phosphodiester bond, removing larger stretch of nucleotides
5) replisome fills gap
6) DNA ligase seals phosphodiester backbone
27
DNA damage and associated repair mechanism
1) direct repair = alkylation
2) BER = depurination / depyrimidination, deamination, oxidative damage
3) NER = UV radiation
28
mismatch repair
- fixes DNA polymerases mistakes (mismatches and small insertions/deletions)
- recognizes newly synthesized strand because of lack of methylation (epigenetic marks copied after complete replication) --> demonstrated in prokaryotic cells, only PREDICTED for eukaryotic cells
29
mismatch repair steps
1) unwind DNA
2) endonucleases cleave phosphodiester bond
3) replisome replaces nucleotides
4) DNA ligase seals phosphodiester backbone
30
xeroderma pigmentosum (XP)
- inherited condition (mutations in XPC and POLH (DNA pol eta))
- extreme sensitivity to UV radiation and risk of UV-induced cancers
- XPC mutations = NER inhibited
- POLH mutations = TLS inhibited = more damage, apoptosis
31
single-strand breaks
- phosphodiester bond breaks on one side of the DNA
- leads to DNA damage response
32
causes of SSBs
1) ionizing radiation: ROS (sugar oxidation) and direct DNA damage
2) inhibition of DNA ligase = unligated Okazaki fragments
3) inhibition of topoisomerase 1 ligation activity
4) stochastic base damage, cytosine demethylation/alkylation, guanine deamination = BER (if fails, leads to SSB)
5) depurination = AP site = beta-elimination (or BER)
33
DNA damage response for SSBs proteins involved
1) lesion identification: PARP1, XRCC1
2) repair of DNA ends: PNKP
3) replace DNA bases:
a) short-patch = pol beta
b) long-patch = pol beta, then epsilon/delta add more, then FEN1 endonuclease removes primers
4) ligation: DNA ligase
34
indirect SSB (BER) DNA damage response
1) detection: APE1/lyase
2) end processing: XRCC1 scaffolds for PNKP and other proteins
3) short-patch filling
4) ligation
35
direct SSB (ex. sugar damage) DNA damage response
1) detection: PARP1/PARG
2) end processing: XRCC1 scaffolds for PNKP and other proteins
3) long-patch filling with PCNA
4) ligation
36
TOP1-SSB DNA damage response
1) detection: RNAP and other lesions
2) end processing: XRCC1 scaffolds for PNKP and other proteins
3) long-patch filling with PCNA
4) ligation
37
double-stranded breaks
- break of phosphodiester bond on both sides of DNA (can be blunt or sticky end)
- more serious than SSB, requiring immediate action
- if not repaired, apoptosis initiated
38
causes of DSBs
1) ionizing radiation
2) ROS
3) type II topoisomerases
4) meiosis: recombination events
5) SSBs during replication: if replication fork encounters SSB, lack of non-covalent interactions can cause replication arm to separate from the fork
39
DSB repair pathways
1) if resection = homology-directed repair (HDR)
2) if no resection = non-homologous end-joining (NHEJ)
40
which DSB repair pathway dominates?
- competitive binding of protein complexes
- if MRN binds first, resection occurs = HDR
- if Ku binds first, resection blocked = NHEJ
41
NHEJ characteristics
- quick and messy
- error-prone
- all phases of cell cycle
42
HDR characteristics
- finding match
- fewer errors
- S and G2 phases
43
HDR types
1) homology between tails = single strand annealing
2) no homology between tails = homologous recombination
44
resection
- removal of some bases around the break to create 3' overhang
45
NHEJ vs HDR protein complexes
- NHEJ = Ku70/80 heterodimer
- HDR = MRN complex
46
NHEJ steps
1) Ku recognizes DSB, acts as scaffold
2) repair proteins recruited
3) ends of chromosome brought together
4) any homology used to reconnect DNA
5) IF NEEDED: gaps filled or excess DNA is removed (pol beta, gamma, mu)
6) backbone ligated together
47
possible outcomes of NHEJ
1) perfect synapsis = no sequence change
2) incorrect microhomology = addition or deletion (indels)
48
HDR (homologous recombination) steps
1) MRN (scaffold) recognizes DSB
2) CtIP recruited, activated by phosphorylation (ATM) and ubiquitylation (BRCA1)
3) MRN/CtIP begins resection: CtIP 5' to 3' endonuclease removes ~100nt
4) Exo1 continues: more efficient (~4kb/h), produces large ssDNA overhangs
5) RPA binds overhangs
6) BRCA2 replaces RPA with RAD51 and paralogs
7) strand invasion: RAD51 coated ssDNA invades dsDNA, searching for homologous region
8) D-loop formed
9) RAD54 removes RAD51, allows DNA polymerase to bind and replicate DNA from template
10) second strand invasion = Double Holiday Junction
11) nucleases resolve junction
49
HDR template options
1) sister chromatid: ideal, because exact same sequence
2) homologous chromosome: could be different allele
3) retrotransposon (homologous sequences): detrimental effects ex. chromosomal translocation and rearrangement
4) artificially introduced repair templates
50
BRCA2 evidence study components
1) long dsDNA with 3' overhang coated with RPA
2) radiolabeled short dsDNA repair template
3) RAD51: same conc. in all samples
4) BRCA2: various conc.
51
BRCA2 evidence study methods
gel electrophoresis:
1) long fragment visible = transfer or repair template = strand invasion occurred
2) short fragment visible = no reaction
52
BRCA2 evidence study results
1) negative control: RPA only = only template
2) positive control: RAD51 only = template and product
3) RPA + RAD51 +increasing BRCA2 conc. = increasing product
53
why are BRCA1/2 mutation associated with increased cancer risk?
- HDR cannot be initiated
- DSBs repaired using NHEJ, which is more error-prone
54
sickle cell anemia
- single amino acid change (Glu6Val) in beta globin gene
- hemoglobin cannot form tetramers, instead form rod-like aggregates that deform RBCs and occlude blood flow
- can be treated with gene therapies
55
genome editing
- alteration of genetic material of living organism by insertion, replacement or deletion of DNA sequence
- typical aim: improve some characteristic of crop/farm animal or correct genetic disorder
56
genome editing exampels
1) site-directed mutagenesis
2) restriction cloning
3) random mutagenesis
4) recombinant DNA cloning
57
recombinant DNA cloning mechanism
1) artificially introduced recombinant DNA molecule triggers DSB repair pathway
2) molecule is homologous to gene of interest, leading to gene replacement
58
recombinant DNA cloning efficiency
- low efficiency
- worked best in organisms that prefer HDR over NHEJ, ex. yeast
59
how to improve genome engineering efficiency
- introduce DSB in genome as well (in target gene)
- end resection occurs on both genome and artificial DNA, allowing for single-strand anealing
60
single-strand annealing
- short-cut for HDR
- homologous tails base pair, flaps are cleaved, DNA ligated
61
which proteins can be used to create DSBs?
1) topoisomerase II
2) restriction enzymes
3) nucleases
4) CRISPR-Cas
62
limitations of topoisomerase, restriction enzymes and engineered nucleases in genome editing
- topoisomerases: cannot target specific sequences
- REs: limited by specific target sequence
- engineered nucleases: require extensive protein engineering
63
types of engineered nucleases
1) zinc-finger nucleases
2) transcription activator-like effector nuclease (TALENs)
64
ZFN vs TALEN
- both have nuclease attached to engineered proteins that recognize DNA sequences
- ZFN: each domain recognizes 3nt
- TALEN: each domain recognizes 1nt
65
what does CRISPR do in bacteria?
1) invasive viral nucleic acids cleaved and incorporated into CRISPR site of bacterial genome
2) expressed as RNA
3) forms complex with endonuclease
4) complex searches for matching viral sequences
5) breaks them = protects against viral infection
66
spCas9
Cas9 from streptococcus pyogenes
67
spCas9 domains
- 2 nuclease domains: RuvC and HNH
- C-terminal domain = PAM-recognition site
- recognition (REC) lobe = sgRNA binding site
- Arg-rich helix = DNA binding
68
spCas9 conformational changes
- apo state: PAM site disordered, nuclease domains collapsed on each other
- sgRNA bound: central channel opens, accommodating dsDNA
69
Cas9 searching
- loaded with guide RNA
- searches dsDNA for PAM site: protein-DNA interaction
- if no site, Cas falls off
70
spCas9 PAM site
5'-NGG-3'
71
Cas9 mechanism
1) dsDNA unwound, stabilized by Arg-rich helix
2) interaction allowed with sgRNA: if enough complementary sequence is found, another conformational change occurs
3) nuclease domains brought into position
4) cleave on both sides to make blunt end: HNH on target strand, RuvC on non-target
72
Cas9 off-target effects
- some mismatch permitted
73
what part of sgRNA has greatest impact on binding?
sgRNA-DNA base-pairing nearest to PAM site
74
what happens after CRISPR-Cas9 cleavage?
- DSB repair or cell death
- NHEJ: can knock-out gene, silent mutation or no mutation
- HDR: can provide repair template for targeted mutation SSA
75
advantages of CRISPR/Cas9
1) targeted
2) sgRNA is cheap and highly efficient (compared to proteins)
3) Cas9 can be modified and/or be a transport protein (dCas9 = no nuclease activity)
76
what proteins can Cas9 transport?
1) nickase: causes SSB (one Cas9 nuclease site mutated)
2) transcriptional regulators
3) chromatin modification: epigenetic modulators
4) tags: ex. GFP
5) base-modifiers
77
applications of CRISPR
1) research: KO models, add tags/domains/modify proteins, change gene expression
2) biotechnology: agriculture and manufacturing
3) healthcare: correct gene mutations, treat disease
78
gene-editing to treat sickle cell anemia
1) screen
2) collect patient stem cell (low risk of rejection)
3) gene-edit, test for function
- turns off expression of BCL11A to allow gamma-globin expression to increase = fetal hemoglobin produced
4) infuse cells
79
ethical concerns around genome editing
1) somatic (adult cells) vs germline (embryos) editing
2) preventing/treating disease vs enhancement
3) risk/benefit profile: availability of reasonable alternatives
4) supporting clinical or pre-clinical data
5) independent ethics reviews
80
CCR5
receptor that HIV binds to get into T cells, may have other important functions that are not completely understood
81
CCR5delta32
- leads to frameshift + premature stop codon
- homozygous individuals for this mutation are resistant to some strains of HIV
- HIV can still infect by interacting with other receptors
82
He experiment (CRISPR babies)
1) CRISPR engineered mutations in CCR5 gene in embryos (mother HIV negative, father HIV positive)
2) no repair template = NHEJ
3) Nana: homozygote with different mutations (1bp insertion, 4bp deletion)
4) Lulu: WT + 15bp deletion (heterozygote)
5) both displayed mosaicism after birth because editing only occurred in some cells of embryo
6) no apparent off-target mutations
83
ethical concerns of He experiment
1) other more effective ways to prevent HIV infection, ex. IVF: remove HIV from sperm first
2) no repair template = cannot guarantee mutation provides resistance
3) debatable informed consent
