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DNA Damage Flashcards

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1
Q

What are the main sources of DNA damage? (7)

A

Any carcinogens
- UV
- Chemical Carcinogens
- Heavy metals
- Reactive metabolites - e.g., reactive oxygen species (free radicals)
- Pollutants
- Ionising radiation
- DNA replication

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2
Q

What are the products of UV induced damage?

A

Pyrimidine dimers - main damage products from UV irradiation
- Formed through cross links between pyrimidine bases (cytosine and thymine)

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3
Q

What are the two main forms of pyrimidine dimers?

A
  • Thymine dimer - cycobutane pyrimidine dimer (CPD)
  • Pyrimidine-pyrimidone 6-4 photoproducts (6-4PPs)
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4
Q

How do CPDs and 6-4PPs affect DNA?

A

Both block progression of inhibit transcription, reduce RNA synthesis and inhibit DNA replication - causing mutagenesis, cell cycle arrest and can cause apoptosis

  • CPDs cause some distortion to the double helix - restored slower (~24hrs) - as products less bulky
  • 6-4PPs twists helix structure - very large helix distortion - restored faster (~4hrs) - bulky products
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5
Q

What is photolyase and why is it important?

A
  • Protein that undoes the thymine dimer
  • Using excited electron from cofactor FADH-
  • Allows cells to survive UV radiation
  • Highly conserved within species - not present in humans
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5
Q

How is the human cryptochrome different without photolyase?

A
  • We have photolyase-like genes - used for circadian rhythm
  • Light sensors (cryptochromes) have same cofactors (flavin - FADH & folate MTHF) but are not active photoproducts
  • So need an alternative pathway to repair UV-induced DNA damage - Nucleotide Excision Repair (NER)
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6
Q

What is the process of Nucleotide Excision Repair (NER)?

A
  1. Detection - damage recognition
  2. Processing - damage removal (helicase unwinding); DNA synthesis (DNA polymerase fill in)
  3. DNA end re-joining - DNA ligation

NER doesn’t reverse damage - it removes it - displacing with new DNA strand

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7
Q

What are the positives and negatives of NER?

A

Positives: low specificity - great power and flexibility (‘catch-all’ repair pathway)
Negatives: is very slow

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8
Q

What is the main disease associated with defects in NER, how was it found and what did it show?

A

Xeroderma Pigmentosum (XP)

  • Genetic Complementation - to find what gene is non-functional in mutant cell lines (transforming mutant cell by re-introducing missing gene to make it wild-type
  • Generate cells, let mutants die; any transformed cells will have NER gene along with mutant; can be easily analysed
  • Found Excision Repair Cross Complementing gene (ERCC) - ‘cross’ because human genes working in rodent cells; yeast mutant gene - RAD
  • Experiments showed that NER pathway is highly conserved in eukaryotes - tested in yeast, rodents, humans
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9
Q

What are the steps of NER in XP?

A
  1. Damage detection - XPC - bulky products detected more easily
  2. Unwinding (helicase); endonuclease complexes cut (XPG;ERCC1/XPF)
  3. Filling in - DNA polymerase then DNA ligase
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10
Q

What happens in NER mutants (XP cells)

A
  • Specialized DNA polymerases - with high error rates
  • Replicate past different damaged bases - translesion DNA synthesis
  • Lack of exonuclease - ‘editing function’
  • E.g., 6-4PPs often lead to base mutations as polymerases are very error prone
  • Highly mutagenic - 100x more likely to get skin cancer
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11
Q

What is transcription-coupled repair (TC-NER)?

A
  • Where DNA damage blocking transcription is specifically removed and the stalled RNA polymerase (RNAP) recruits the NER machinery
  • CSA & CSB - (CSB binds to blocked polymerase - recruits repair factors to help RNAP move past damage)
  • RNA polymerase detects blocking forms of DNA damage - instead of recognition proteins
  • TC-NER bypasses need for XPC damage recognition
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12
Q

How do the photoreactivation and NER pathways compare?

A

Photoreactivation
- Single enzyme - photolyase
- Light-dependent reversal of UV-induced regions
- Used light energy between 300-500nm
- Limited substrate range

NER
- Lots of proteins involved
- Multi-step process
- Broad range of substrates - e.g., pyrimidine dimers
- Light-independent reduction of UV-induced regions (not reversal)

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13
Q

What are the various repair pathways and which ones work on one strand (SSBs) /broken chromosomes (DSBs)?

A

One strand - use other as template:
- Direct reversal - e.g., photolyase/methylguanine transferase
- Mismatch Repair (MMR) - main function isnt DNA repair
- Nucleotide Excision Repair (NER) - catch all pathway - UV - pyrimidine dimers
- Base Excision Repair (BER) - very active pathway - specific substrates

Work on broken chromosomes:
- Homologous recombindation (HR)
- Non-homologous end joining (NHEJ)

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14
Q

How can you test for a mutagen?

A

Ames test

  • Relies on mutation of non-functional histidine biosynthesis gene
  • Certain mutations will cause cause a mutation that restores the function of the gene
  • Bacteria on histidine-deficient media are counted
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15
Q

What causes the most DNA damage events in cells?

A

Reactive Oxygen Species
- Damage bases - and can also damage sugar-phosphate backbone
- Most common damage - guanine - 8-oxoguanine (8-oxo-G) - mutation events occur during S phase

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16
Q

How is deamination mutagenic?

A

Deamination (loss of amino group) from cytosine converts to uracil - which is mutagenic in DNA
- As when DNA polymerase passes Uracil - it deposits adenine rather than guanine (if cytosine was still there)

Important feature of DNA damage: damage is either irreversibly fixed, or it is made considerably worse by passing through cell cycle (e.g., S phase - DNA replication or mitosis (chromatid separation and segregation into daugher cells)

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17
Q

How can cytosine deamination be exacerbated by epigenetic processes and how is it detected?

A
  • Cytosine is normally found in eukarotic cells in form of 5-methy cytosine - from epigenetic regulation
  • Oxidative deamination of 5-methyl-C produces thymine
  • However this thymine is bound to guanine (from cytosine deamination to uracil) and so it is a base pairing of T:G
  • Detected by mismatch glycosylase - distinct to MMR - a specialized case that corrects errors in replication due to to nucleotide misincorporation
  • Most MMR doesn’t involve glycosylases - and uses diff pathway
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18
Q

What is the role of MMR, and how does it work (briefly)?

A
  • Important in DNA maintenance, repair, recombination and replication
  • Doesn’t use BER or glycosylases
  • Mismatch complexes are used - MSH2-MHS6 heterodimers bind to single base-pair mismatches
  • Mutations in MHS2/MHS6 - predisposition to non polyposis colon cancer (HNPCC)
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19
Q

What is Base Excision Repair, and what is the process?

A

BER - general repair pathway for DNA single strand breaks

  1. Detection - initiated when damaged base is removed by glycosylase or spontaneously (depurination) - leaving abasic site
  2. Processing - sugar phosphate backbone is removed; is replaced - new DNA synthesis - short/long patch repair
  3. DNA end re-joining - ligase
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20
Q

What is difference between short/long patch BER?

A

Short patch BER:
- Cut backbone either side of abasic site (AP lyase & AP endonuclease); backbone is cut again - so small fragment can be removed - creates intermediate that is a SS DNA break - which is then repaired (PARP)

Long patch BER:
- Nick created by AP endonuclease allows polymerase to start filling in - displacing/replacing nucleotides in a longer patch (2-10 nucleotides)

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21
Q

How is alkylation relevant to DNA damage - and what is responsible for donating methyl groups?

A
  • Very common - e.g., DNA methylation (type of alkylation)
  • S-adenosyl methionine (SAM) donates methyl group to cytosine - to produce epigenetic mark (5-methyl-C) - but SAM can also add methyl group to non-target bases - e.g., 3-methyl adenine (blocks DNA replication)
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22
Q

How has cytosine been lost over evolution in the context of CpG islands?

A
  • C is usually methylated (5-methyl-C)
  • Deamination converts it to T is not fully reversed by thymine DNA glycosylase activity - leading to loss of C over time
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23
Q

Why is 5-methy-C a problem in the genome in relation to UVB irradiation?

A
  • When C is methylated - it forms CPDs (UV) more readily than the unmodified base
  • Made worse - when in form of a CPD - amino group of 5meC is more readily lost - making C-T transitions a major consequence of UVB irraditation
  • Made even worse - when by-pass DNA polymerases that synthesise DNA using CPD template typically insert an ‘A’ opposite the CPD - later this ‘A’ is used in the template and replaced by T residues …
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24
Q

What is the significance of Mitomycin C?

A
  • Alkylating agent with two active alkylation sites that can bond with two different DNA strands; linking strands - making DNA replication impossible
  • Very toxic compounds to dividing cells - used as chemotherapy reagant - as cancer cells race through cell cycle checkpoints - making them hypersensitive to DNA damage
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25
Q

What problems does ionising radiation cause for DNA?

A
  • Interact with water - creating free radicals (ROS)
  • High local concentrations - which can cause DS breaks (most toxic); are much harder to repair - one DS break can kill a cell/cause cancer
  • DS breaks repaired by recombination - joining of two DNA molecules - but broken ends may not remain juxtaposed; can cause chromosome fusions, degradation and loss of genetic material
  • Whereas SS break - keeps duplex intact
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26
Q

What are the two pathways for repairing DS DNA breaks?

A

1. Non-homologous end-joining (NHEJ) / IR
- DNA ends are joined - largely independent of their sequence - errors (short insertions/deletions) can occur

2. Homologous recombination (HR) - RAD51 pathway
- Uses intact copy of damaged region as template for repair
- This is usually a sister chromatid, but can also be a homologous chromosome or somewhere else in the genome

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27
Q

When are NHEJ and HR used in yeast/mammals?

A

Yeast - tend to repair DSBs via HR

Mammals - preferentially repair DSBs via NHEJ (especially in G1) - but HR is very important in S phase
- NHEJ enzymes out-compete HR enzymes for DSB joining

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28
Q

What is the definition of recombination?

A

The joining together of two DNA duplexes to create a new molecule

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29
Q

Why are BER and NER pathways both required?

A
  • Detection of SSB’s - by PARP recruits BER factors PolB, Lig3 and XRCC1
  • NER can’t repair a mis coded base like 8-oxo-G very well
  • BER can’t repair CPDs
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30
Q

What are the sources of DSBs?

A
  • High energy radiation - create localized areas of ROS (that simultaneously attack both strands)
  • DNA replication damage
  • Topoisomerase failures
  • Mechanical stress
  • Inadvertant enzyme action
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31
Q

How can DNA replication cause DSBs?

A
  • Torsional stress as helix is unwound - topoisomerases relax coiled DNA
  • Replication fork encounters problems - DNA damage (photoproduct/5-methly-C) - preventing progression - or can replicate across; SSB (if not repaired by BER/NER)
  • This can lead to a free DS DNA end
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32
Q

What is translesion synthesis, what do they use and what are the issues associated with it?

A

Translesion synthesis is replication past damaged bases - using translesion polymerases - regulated through PCNA

  • But have lower fidelity and make many error
  • Lack of exonuclease with ‘editing function’
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33
Q

What experiment was used to identify NHEJ genes, and what genes are they?

A
  • X-ray cross complementing (XRCC) - hamsters transfected with human genes; identified human gene that are repair competent of x-ray induced DNA damage
  • XRCC(4-7) as NHEJ genes
  • XRCC5 (human KU80) rescues xrs-6 mutants (that are hypersensitive to x-rays) - XRCC5 returns mice to wild-type sensitivity
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34
Q

How does XRCC5/KU80 restore wild-type x-ray sensitivity in Xrs-6 CHO cells?

A
  • Competition of cut plasmids vs uncut plasmids showed that mutant is lacking a protein that is specific for binding DNA ends
  • XRCC5 (KU80) forms (dimer) with XRCC6 (KU70)
  • Ku complex binds DNA ends (forms ring) - preventing excessive degradation; facilitates repair and protien recruitment
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35
Q

Main steps of NHEJ

A
  1. Synapsis (bringing together) - DNA ends are rapidly synapsed - suppressing chromosome translocations
  2. This forms long-range synaptic complex - Ku complex with a protein kinase DNA-dependent protein kinase catalytic subunit (only found in vertebrates) to form complex: DNA-dependent protein kinase (DNA-PKcs)
  3. Additional DNA end processing factors
  4. Final ligation step - required for short-range synaptic complex - catalysed by specific DNA ligase termed LIG4 (recurited through XRCC4) LIG4-XRCC4
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36
Q

What two other key nucleases does DNA-PK phosphorylate? (Outside this topic)

A
  • Artemis - ability to process DNA hairpins (V(D)J recombination - NHEJ)
  • H2AX - important in signalling the presence of DSB to the cell; can extend for 10’s of thousands of bases either side of DSB. After processing, DSB can be ligated by LIG4 in complex with XRCC4
37
Q

Many enzymatic activities may be required at the site of a DSB, but what diseases can be associated with mutations in these genes?

A

Werner Syndrome (WRN) / premature ageing

38
Q

How is NHEJ different to say BER or NER?

A
  • Works on DSBs
  • Less linear pathway - lots of redundancy built into the system - providing the ability to cope with unexpected forms of damage
  • Depending on nature of DNA damage - a variety of enzymes can be recruited - (Recruited by Ku Complex)
39
Q

What is alternative end-joining (Alt-EJ) and how does it link to NHEJ?

A
  • Ku independent - alternative pathways for repairing DSBs
  • PARP recruits SSB repair factors
  • Removes any core NHEJ factors; uses micro homologies in end-joining (MMEJ)
  • More active in cells that lack Ku - but, ends are more prone to nuclease digestion in absence of Ku
  • Pol-theta - lack of has shown little/no end joining
40
Q

What is backup-NHEJ (B-HEJ)?

A
  • Separate Ku-independent pathway
  • Borrows some factors from SS DNA break repair / short-patch BER
41
Q

How is ATM important signalling DNA damage?

A
  • ATM phosphorylates 1000s of proteins, signals DNA damage leading to activation of repair, cell cycle arrest and cell death
  • Activated by MRN complex; interacts with NBS1 component
  • Within minutes - ATM phosphorylates cellular targets - e.g., H2AX - which signals damage to the cell
42
Q

In what context is HR particulary useful?

A
  • When DNA replication causes a DS break (and in meiosis)
  • Can’t be fixed by NHEJ because it hasn’t been repliacted yet - nothing to join it with
  • Need a pathway to rebuild replication fork - HR
43
Q

What are the three pathways in homologous recombination (HR)?

A
  1. Synthesis-dependent strand annealing (SDSA)
  2. Double-strand break repair model (DSBR)
  3. Single strand annealing (SSA) - only if the break occurs between repeats
44
Q

When/how does single strand annealing (SSA) work?

A
  • Only if break occurs between repeats
  • Resection allows SS region to anneal - compementary regions
  • Repairs DSB - intervening sequence is deleted
  • Doesn’t require central HR factors - e.g., RAD51
  • Fairly frequent - as lots of repeated sequences in genome
  • But makes genome shorter - due to deletion
45
Q

When/how does synthesis-dependent strand annealing (SDSA) work?

A
  • Most common
    1. DSB detection and recruitment of HR proteins
    2. Resection to produce 3’ tails
    3. Homology search to find region of similarity - RAD51 - uses it as a template
    4. Strand invasion
    5. DNA synthesis - using template strand - therefore displaces non-template strand of the invaded duplex - forming a displacement loop (D loop)
    6. Re-annealing; DNA synthesis and DNA ligation completes the strand

MMR checks strand & results in gene conversion

46
Q

What is meant by ‘resection’ in the context of a DSB? And what is it useful for?

A
  • In DSBs - breaks are first processed by nucleolytic enzymes to produce long 3’ SS DNA tails - called ‘resection’
  • These SS tails are the substrate for the homology search
47
Q

What is RAD51 and what is its role?

A
  • Recombinase protein - catalyses recombination
  • Forms nucleoprotein filament with SS DNA that invades the duplex
  • Capable of finding sequence homology in an entire genome
  • Protected by replication protein A (RPA)
48
Q

Explain the double-strand break repair model (DSBR)

A
  1. DSB detection and recruitment of HR proteins
  2. Resection to produce 3’ tails
  3. Homology search - RAD51
  4. Strand invasion displacing non-template strand DNA - forms D-loop
  5. Other end of DNA interacts with D-loop - forming a double holiday junction
  6. Crossing over can occur between strands - meiosis - genetic diversity - sister chromatid splitting
49
Q

How are SS DNA tails generated for HR?

A
  1. DSB - detection
  2. Removal of blocked ends - MRN/CtIP - MRE11 (endonuclease activity - with help from CtIP - chops oligonucleotide off end of DNA)
  3. Resection: Helicase (BLM in protein complex) / nuclease - DNA2/EXO1 - digest ends - to make long highly recombinagenic SS DNA region
  4. Binding of ss DNA by RPA - stabilises/protects
50
Q

Why can HR be dangerous in mammals?

A
  • Animal genomes are full of repetitive DNA
  • So if RAD51 was let loose in all cells - it woulf scramble our genomes through recombination - dangerous
  • Can happen in some cancer cells
51
Q

How is HR regulated in the cell cycle?

A
  • G1 - HR is downregulated - as is no nearby sister chromatid - if breakage occured in a repetitive region (Alu element) - recombination could occur - but not bad as long as there isnt crossing over
  • G2 - upregulation of HR chromatids are aligned in cell - facilitating HR
  • S-phase - HR is very important - main way to fix a broken replication fork
52
Q

How is HR regulated in humans?

A
  • Breast cancer type 2 susceptibility protein (BRCA2) controls activity of RAD51
  • Controlling activity of main recombinase RAD51 - controls HR
53
Q

What is BRCA2 and how does it control RAD51?

A

BRCA2 is a tumour suppressor gene that regulates RAD51 activity - allows HR to take place

  • Prevents unwanted RAD51 DNA binding
  • Recruits RAD51 to ssDNA tails
  • Helps displace RPA and load RAD51
  • Reduces RAD51 off-loading
54
Q

What are PARP inhibitors, and what do they do?

A

PARP inhibitors (Poly(ADP-ribosyl) polymerase
- They prevent SSB repair - creating more DS breaks - thus needing HR
- Some cancers have deficient HR so - can be used to treat cancer - killing BRCA deficient cells

55
Q

What blocks DNA ends in G1 to prevent HR?

A
  • 53BP1blocks DNA ends in G1
  • Prevents resection
56
Q

How is CtIP activated in G2?

A

CtIP is inactivated in G1 and activated in G2 by cyclin dependent kinase (CDK)

-Phosphorylation of CtIP on serine 327 acts as a cell-cycle-dependent switch that regulates CtIP-dependent DNA end resection

57
Q

How does BRCA1 stimulate HR?

A
  • Blocks promotes 53BP1 removal
  • Promotes BRCA2 loading of RAD51 onto DNA (PALB2)
  • Increases CtIP resection
58
Q

How does mutation of BRCA1 cause cancer?

A
  • Mutations in BRCA1 cause genomic instability - through misregulation of recombination pathways - control HR
  • Mutations - ~50% chance of ovarian cancer by age of 70
59
Q

When are SDSA and DSBR pathways chosen? And what syndrome is associated with promoting SDSA?

A
  • Most cells we dont want cross over - so we want SDSA NOT DSBR
  • BLM bloom syndrome helicase (BLM) promotes SDSA - by disrupting D-loops that prevent crossing-over - unwinds invading strand
  • BLM prevents formation of double-holiday junction
60
Q

How is crossing over determined in DSBR?

A
  • Holiday junction resolution by holiday junction resolvases will determine whether crossing over occurs
  • One mechanism - endonuclease - Gen1
61
Q

What is Gene Targeting (GT)?

A
  • Gene insertion or modification at a specific site in the genome
  • Generation of specific alterations in a genome by HR-mediated integration of foreign DNA sequences
62
Q

What is Gene Targeting (GT) dependent on?

A

GT is dependent on homology dependent repair (HDR) / integration of a transgene
- Relies on HR for HDR

63
Q

What is Gene Editing (GE)?

A
  • Site-directed modification of genes including mutations and precise changes to sequences
64
Q

What various technologies have been developed that make specific breaks in the genome?

A
  • Homing endonucleases
  • Zinc-fingered nucleases (ZFNs)
  • TALENs
  • CRISPRs
65
Q

Why is GT more challenging in mice/humans than yeast?

A
  • Yeast - lots of HR - so can PCR amplify a marker gene with 35-40 bases of sequence homology to gene of interest - triggers Homologous Recombination (HR)
  • Humans/mice - several factors are necessary for successful GT in mice
  • Mostly do NHEJ - so need to promote HR
  • AND - length of homology required is much greater than yeast
66
Q

How was GT developed in mice?

A

Discovered that long tracts (up to 10kb) of homologous sequence is not enough to drive GT in mice

So, used:
- Used Negative Selection - works by having a gene - that if present causes cell death
- Insert marker gene flanking the transgene - outside the region of homology
- So - if HR occurs at transgene - it results in loss of this marker
- Whereas if the transgene integrates at a random site in the genome - the marker is integrated - and these cells are killed (thymidine kinase - herpes)
- Using embyronic stem cells - support high levels high rates of GT - taken from fertilised embryos

67
Q

What are homing endonucleases and how did they improve GT in mice?

A
  • Specific endonucleases that cut DNA at a specific place - create DSB
  • Are parasitic DNA - like transposons - and so are replicating DNA elements - but do not encode enzymes or cut/paste themselves into the genome
  • Allows DSBs to be introduced into certain sites in a controlled way
  • Induction of DSB enhanced GT frequency by 50 fold in embryonic stem cell mice experiment
68
Q

What are the limitations of homing endonucleases?

A
  • Recognition site has to be engineered into the genome in the first place
  • Limits options for genome editing - as transgenes are only introduced at engineered sites with endonuclease recognition sequence
  • Thus, endonucleases cannot be used to target endogenous genes
69
Q

What are zinc-fingered transcription factors?

A

Transcription factors made up of two domains:
- DNA binding and transcriptional activation
- Alpha-helix and two beta sheets with zinc ion
- E.g., CysHis2 zinc finger - 28-30 AAs
- Each finger recognises principally 3 bases pairs of DNA through contact with major groove

70
Q

What is the Fokl enzyme and why is it important?

A
  • Fokl restriction endonuclease - distinct module from DNA recognition domain
  • This cleavage domain is used for construction of ZFNs
  • Monomers are inactive - and two nuclease domains must dimerise for the enzyme to have activity
  • Helps generate specificity for the endonuclease
71
Q

Structure and function of a zinc-fingered endonuclease (ZFN)

A

Two components:

  • Endonuclease enzyme from restriction enzyme Fokl - fused to a DNA recognition domain made up from an array of zinc finger DNA binding domains

Function:
- Recognition of specific sequences allows enzymes to introduce breaks at target locus
- Can be used to generate mutations in genes or stimulate target integration of a transgene

72
Q

What are the limitations of ZFNs?

A
  • Very difficult ot make - error rate in lab is very high at each stage
  • Beyond reach of most labs
73
Q

How could ZFNs be used to cure genetic diseases in the future?

A
  • Integrate functional copy of gene and remove problems caused by random insertion - true gene correction
  • ‘proof of principle’ has been carried out - in mutated ILR2G gene
  • Targeting was found in around 20% of cells
  • But integration of gene was not analysed
  • Could be promising in future - as quality of protein engineering and ZFNs improves
74
Q

What are TALE nucleases (TALENs) and how do they work?

A
  • Transcription activator-like effector nucleases
  • Based around proteins that are synthesised by bacteria - a plant pathogen - bacteria has developed a system that helps infection of the plant - secretion of protein into plant cell
  • DNA binding domain, transcriptional activation
  • Approach to develop endonucleases is same as ZFNs - DNA binding module + Folk endonuclease
  • Used in pigs - genetically suppress growth hormone receptor - micro pigs
75
Q

Positives/limitations of TALENs?

A

Positives:
- Highly effective
- Far more predictable about how they bind - scientists can target exactly where they want

Limitations:
- Like ZFNs - TALENs are very complex to synthesise
- Require assembley of DNA fragments to produce full length gene
- Repetitive nature of these genes can lead to instability

76
Q

What is the CRISPR/Cas9 system and how does it work?

A

Based on bacterial defence:
- Bacteria take short segment of invading DNA (e.g., virus/bacteriophage) and use this to guide an endonuclease to create DSBs in any similar sequences
- This segment of foreign DNA is inserted into a repetitive array - that leads to expression of foreign segment as part of large RNA molecule (CRISPR)
- This RNA isassociated with Cas9 enzyme - and foreign segment is positioned to guide the Cas9 to the target
- Endonuclease breaks can be used for mutagenesis or HDR (homology directed repair)
- Further reading - e.g., cure for CTFR
*
- CRISPR region - repeated region
- Cas9 - CRISPR associated endonuclease*

77
Q

What recent methods has CRISPR been used for that has had controversy?

A

CRISPR Gene Therpay
- Engineering people - in germline
- Gene edited babies - to make them resistant to say HIV or CF
- Big ethical issues
- Further reading

78
Q

What two ways may a cell deliberately induce DSBs?

A
  • In meiosis - genetic diversity - crossing over
  • V(D)J recombination - class switch recombination - to generate antibody diversity when V(D)J regions rejon
79
Q

Recap of mitosis vs meiosis - including key role of prophase/metaphase

A

Mitosis - 4n diploid cell to 2n single chromatids (no interaction between homologous chromosomes)
- Prophase - chromosomes condense
- Metaphase - chromosomes align on mitotic spindle

Meosis - 4n to n (haploid) gametes
- Prophase 1 - homologous chromosomes pair up (crossing over - at least one event needed)
Meiosis 2 - same as mitosis

80
Q

How does meiosis differ in males/females?

A
  • Male - very quick
  • Females - much slower - can cause problems - older oocytes accumulate mutation
  • In part - due to cohesion of sister chromatids over time
81
Q

What is chromosome non-disjunction, and what can it cause?

A
  • Includes addition of additional chromosomes or loss of one (aneuploidy)
  • Aneuploidy is major cause of infertility, miscarriage and congenital birth defects
82
Q

What are bivalents, and when do they form?

A

Homologus pair of chromosomes = bivalent

  • Highly regulated process - needs one crossover (prophase 1)
  • Doesn’t want too many (1/2) - crossover inteference/assurance
83
Q

How is HR initiated in meiosis?

A
  • Spo11 protein (topoisomerase) makes DSBs
  • But differently to other topoisomerases it sits covalently bonded to end of that break - creating blocked end - Spo11 may prevent NHEJ??
  • MRE11/CtIP needed to remove blocked end (spo11)
  • Exo1 or BLM-DNA-2 complexes - both able to generate long 3’ tails to intiate recombination
84
Q

Why and how do we promote DBSR in meiosis?

A
  • Want the opposite to normal - to promote formation of crossover products
  • DMC1 - recombinase similar to RAD51 - but only functions in meiosis
85
Q

What is DMC1 and why is it important?

A
  • DMC1 - promotes crossovers in meiotic recombination
  • DMC1 forms search tentacle - goes looking for homologous sequence - nucleoprotein filament
  • DMC1/RAD51 are loaded differently on 3’ DNA ends - discussion here - no conclusive evidence
  • DMC1 formed D-loops are more resistant to (BLM) helicase disruption than RAD51 - so, effectively BLM is down-regulated and RAD51 is inhibited in meiosis
  • HOP2-MDN1 complex stimulates DMC1 acitivity and stabilises DMC1-ssDNA nucleoprotein complexes + facilitates conjoining of DNA molecules through capture of dsDNA by DMC1-ssDNA nucleoprotein filament
86
Q

What is the synaptonemal complex (SC) in meiosis?

A
  • Tripartite proteinaceous structure (SC) promotes interhomologue recombination
  • Determines outcome of DSB- whether crossing over or not
  • Regulates location of crossovers
87
Q

What DNA sequence motifs are associated with crossovers?

A
  • Loose DNA sequence motifs
  • Methylation of lysine 4; acetylation of lysine 9 of histone 3
88
Q

How can you reduce/promote crossovers in mitotic/meiotic cells?

A

Mitotic:
- RAD51 controlled by BRCA2 - BLM helicase promotes SDSA model - which unwinds D-loop - removing holiday junction
- HR is tightly regulated - active in S-phase and G2

Meiotic:
- DMC1 filaments are resistant to BLM helicase - promoting DSBR model
- DMC1 stabilises D-loops (HOP2-MND1 complex) - forms holiday junctions
- Even so - crossovers are still low (1-3 per chromosome pair) whereas no. of DSBs created is far greater (10-40 fold)

89
Q
A

Advanced Genetics (4 decks)

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