DNA Damage Flashcards
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
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)
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)
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
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
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)
6
Q
What is the process of Nucleotide Excision Repair (NER)?
A
- Detection - damage recognition
- Processing - damage removal (helicase unwinding); DNA synthesis (DNA polymerase fill in)
- DNA end re-joining - DNA ligation
NER doesn’t reverse damage - it removes it - displacing with new DNA strand
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
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
9
Q
What are the steps of NER in XP?
A
- Damage detection - XPC - bulky products detected more easily
- Unwinding (helicase); endonuclease complexes cut (XPG;ERCC1/XPF)
- Filling in - DNA polymerase then DNA ligase
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
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
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)
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)
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
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
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)
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
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)
19
Q
What is Base Excision Repair, and what is the process?
A
BER - general repair pathway for DNA single strand breaks
- Detection - initiated when damaged base is removed by glycosylase or spontaneously (depurination) - leaving abasic site
- Processing - sugar phosphate backbone is removed; is replaced - new DNA synthesis - short/long patch repair
- DNA end re-joining - ligase
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)
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)
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
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 …
24
What is the significance of Mitomycin C?
- 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
25
What problems does ionising radiation cause for DNA?
- 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
26
What are the two pathways for repairing DS DNA breaks?
**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
27
When are NHEJ and HR used in yeast/mammals?
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
28
What is the definition of recombination?
The joining together of two DNA duplexes to create a new molecule
29
Why are BER and NER pathways both required?
- 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
30
What are the sources of DSBs?
- High energy radiation - create localized areas of ROS (that simultaneously attack both strands)
- DNA replication damage
- Topoisomerase failures
- Mechanical stress
- Inadvertant enzyme action
31
How can DNA replication cause DSBs?
- 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
32
What is translesion synthesis, what do they use and what are the issues associated with it?
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'
33
What experiment was used to identify NHEJ genes, and what genes are they?
- **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
34
How does XRCC5/KU80 restore wild-type x-ray sensitivity in Xrs-6 CHO cells?
- 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
35
Main steps of NHEJ
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
36
What two other key nucleases does DNA-PK phosphorylate? (Outside this topic)
- 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
Many enzymatic activities may be required at the site of a DSB, but what diseases can be associated with mutations in these genes?
Werner Syndrome (WRN) / premature ageing
38
How is NHEJ different to say BER or NER?
- 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
What is alternative end-joining (Alt-EJ) and how does it link to NHEJ?
- 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
What is backup-NHEJ (B-HEJ)?
- Separate Ku-independent pathway
- Borrows some factors from SS DNA break repair / short-patch BER
41
How is ATM important signalling DNA damage?
- 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
In what context is HR particulary useful?
- 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
What are the three pathways in homologous recombination (HR)?
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
When/how does single strand annealing (SSA) work?
- 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
When/how does synthesis-dependent strand annealing (SDSA) work?
- 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
What is meant by 'resection' in the context of a DSB? And what is it useful for?
- 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
What is RAD51 and what is its role?
- 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
Explain the double-strand break repair model (DSBR)
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
How are SS DNA tails generated for HR?
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
Why can HR be dangerous in mammals?
- 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
How is HR regulated in the cell cycle?
- **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
How is HR regulated in humans?
- Breast cancer type 2 susceptibility protein (BRCA2) controls activity of RAD51
- Controlling activity of main recombinase RAD51 - controls HR
53
What is BRCA2 and how does it control RAD51?
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
What are PARP inhibitors, and what do they do?
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
What blocks DNA ends in G1 to prevent HR?
- 53BP1blocks DNA ends in G1
- Prevents resection
56
How is CtIP activated in G2?
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
How does BRCA1 stimulate HR?
- Blocks promotes 53BP1 removal
- Promotes BRCA2 loading of RAD51 onto DNA (PALB2)
- Increases CtIP resection
58
How does mutation of BRCA1 cause cancer?
- Mutations in BRCA1 cause genomic instability - through misregulation of recombination pathways - control HR
- Mutations - ~50% chance of ovarian cancer by age of 70
59
When are SDSA and DSBR pathways chosen? And what syndrome is associated with promoting SDSA?
- 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
How is crossing over determined in DSBR?
- Holiday junction resolution by holiday junction resolvases will determine whether crossing over occurs
- One mechanism - endonuclease - Gen1
61
What is Gene Targeting (GT)?
- 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
What is Gene Targeting (GT) dependent on?
GT is dependent on homology dependent repair (HDR) / integration of a transgene
- Relies on HR for HDR
63
What is Gene Editing (GE)?
- Site-directed modification of genes including mutations and precise changes to sequences
64
What various technologies have been developed that make specific breaks in the genome?
- Homing endonucleases
- Zinc-fingered nucleases (ZFNs)
- TALENs
- CRISPRs
65
Why is GT more challenging in mice/humans than yeast?
- **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
How was GT developed in mice?
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
What are homing endonucleases and how did they improve GT in mice?
- 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
What are the limitations of homing endonucleases?
- 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
What are zinc-fingered transcription factors?
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
What is the Fokl enzyme and why is it important?
- **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
Structure and function of a zinc-fingered endonuclease (ZFN)
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
What are the limitations of ZFNs?
- Very difficult ot make - error rate in lab is very high at each stage
- Beyond reach of most labs
73
How could ZFNs be used to cure genetic diseases in the future?
- 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
What are TALE nucleases (TALENs) and how do they work?
- **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
Positives/limitations of TALENs?
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
What is the CRISPR/Cas9 system and how does it work?
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
What recent methods has CRISPR been used for that has had controversy?
**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
What two ways may a cell deliberately induce DSBs?
- **In meiosis** - genetic diversity - crossing over
- V(D)J recombination - **class switch recombination** - to generate antibody diversity when V(D)J regions rejon
79
Recap of mitosis vs meiosis - including key role of prophase/metaphase
**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
How does meiosis differ in males/females?
- Male - very quick
- Females - much slower - can cause problems - older oocytes accumulate mutation
- In part - due to cohesion of sister chromatids over time
81
What is chromosome non-disjunction, and what can it cause?
- Includes addition of additional chromosomes or loss of one (aneuploidy)
- Aneuploidy is major cause of infertility, miscarriage and congenital birth defects
82
What are bivalents, and when do they form?
**Homologus pair of chromosomes** = bivalent
- Highly regulated process - needs one crossover (prophase 1)
- Doesn't want too many (1/2) - crossover inteference/assurance
83
How is HR initiated in meiosis?
- **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
Why and how do we promote DBSR in meiosis?
- Want the opposite to normal - to promote formation of crossover products
- DMC1 - recombinase similar to RAD51 - but only functions in meiosis
85
What is DMC1 and why is it important?
- 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
What is the synaptonemal complex (SC) in meiosis?
- Tripartite proteinaceous structure (SC) **promotes interhomologue recombination**
- Determines outcome of DSB- whether crossing over or not
- **Regulates location of crossovers**
87
What DNA sequence motifs are associated with crossovers?
- Loose DNA sequence motifs
- Methylation of lysine 4; acetylation of lysine 9 of histone 3
88
How can you reduce/promote crossovers in mitotic/meiotic cells?
**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
