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DSB 3 Flashcards

(40 cards)

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1
Q

What is the main difference between HR and NHEJ?

A

HR requires a repair template

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2
Q

When does HR occur?

A

In late S/G2 when there is a sister chromatid (not homologous chromosome)

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3
Q

When are sister chromatids not used in HR?

A

During meiosis

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4
Q

Describe the general HR pathway

A
  1. Break is recognize
  2. Remove some bases from either side of the break to make some stretches of ss DNA (DNA resection)
  3. Enzymes bind to ss DNA = strand exchange reaction where there is base pairing with repair template chromatid
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5
Q

What is the role of the enzymes at the ends of the broken strands in HR?

A

To find a piece of DNA that is identical to the missing piece

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6
Q

What are the 4 models for DSB repair that require HR?

A
  • Double Holiday Junction model (DSBR)
  • Synthesis dependent strand annealing (SDSA)
  • Break induced replication (BIR)
  • Single strand annealing (SSA)
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7
Q

Why can HR be mutagenic even though we use an identical repair template?

A

Because SSA can lead to large deletions

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8
Q

In what direction does DNA resection occur?

A

5’ to 3’ direction. Therefore, the 3’ end is the one that is exposed at the end of the break.

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9
Q

Why is the 3’ end so important?

A

Because 3’ end is what is used by DNA pol to extend. No pols can extend the 5’ end.

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10
Q

Describe the HR pathway is detail

A
  1. After break, HR is initiated using MRN complex and CtiP
  2. We then create long range ss DNA via Exo1 (bases are removed)
  3. RPA coats the ssDNA which protects the fragility of the ssDNA ends and promotes loading of next repair factors like RAD51
  4. RAD51 replaces RPA, mediated by RAD52 and BRCA2 = formation of helical DNA protein filament (nucleoproteinn filament)
  5. The RAD51 coated ss invades onto the duplex DNA (the good sister chromatid strands) and displaces one strand = displacement loop
  6. The 3’ end of the invading strand is tested for homology and if everything looks good it is extended by pol, copying the template strand
  7. The second end of the DSB joins with the D loop by RAD52.
  8. Then DNA synthesis occurs via pol to fill gaps of the 4 strands = Double holiday junction
  9. Helicases like BLM will push the junctions towards each other = hemicatenane
  10. Topoisomerase nicks the hemicatenane and it is then religated so strands move back, this is dissolution = Noncrossover recombinant products
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11
Q

Why is it dangerous to expose lots of ssDNA?

A
  • ssDNA is very fragile so can get nicked and fall apart easily via things like free radicals
  • The longer the stretch of ssDNA, the more potential it has to find pseudohomology = translocations
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12
Q

Why does HR use the sister chromatid?

A

Because it is super close and it is identical obvs

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13
Q

What is heteroduplex DNA?

A

This is when 2 strands of DNA in the holiday junction are are derived from different DNA molecules.

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14
Q

Why is the holiday junction a thermodynamic structure?

A
  • It can move from left to right without using any energy

- It can also be pushed by enzymes like helicases (BLM/WRN) that migrate molecules = holiday junction migration

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15
Q

When do heteroduplex DNAs arise?

A

This arises when a holiday junction occurs between non homologous DNA strands, therefore there is not perfect base pairing.

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16
Q

What happens as a result of heteroduplex DNA?

A

Either mismatch repair or inherited as mutation

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17
Q

How do we get rid of the holiday junction once we are done?

Study These Flashcards
A

Cleave it and migrate the strands into place properly, this is called resolution.

18
Q

What is the difference between a hemicatenane and a full catenane?

Study These Flashcards
A

Full: the full duplex is intertwined

Hemi: 2 of the ss from separate molecule is intertwined

19
Q

What is the difference between crossovers and noncrossovers?

Study These Flashcards
A

CO: reciprocal exchange of flanking markers (only involved in resolution)

NCO: non reciprocal exchange of flanking markers (involved in dissolution and resolution)

20
Q

When is crossover essential and repressed?

Study These Flashcards
A
  • Essential in meiosis

- Repressed in somatic cells

21
Q

What is the difference between dissolution and resolution?

Study These Flashcards
A

Dissolution: used topoisomerases to catalyze decatenation, this gives rise to NCO recombinant products so there’s no exchange and no heterozygosity between homologous chromosomes.

Resolution: catalyzed by endonucleases (resolvases) that cleave the junction.

22
Q

What are the classic resolvases?

Study These Flashcards
A
  • Gen 1 in mammalian cells
  • Yen1 in yeast
  • Othologues of RuvC (classic bacterial resolvase)
23
Q

What is synthesis dependent strand annealing (SDSA)?

Another repair process that requires HR

Study These Flashcards
A
  • Second side of the break never gets involved in recombination.
  • Invaded end gets displaced and just re-anneals with the second side of break.

= creates non crossover products as well

24
Q

What is break induced replication? BIR

Another process for repair that requires HR

Study These Flashcards
A

Break gets resected after recognition, then one side of break invades and then replication just continues without annealing until end of molecule is reached

25
What is single strand annealing? SSA Another process for repair that requires HR
1. Recognition 2. SS resection uncovers direct repeats 3. So these amazing direct repeat sequences that are single stranded can anneal with each other leaving some excess flaps 4. Flaps are trimmed off by flap endonuclease (Fen1) 5. Pol fills gaps and all just ligated together
26
What happens if SSA occurs in the middle of a gene (between non homologous shit)? Don't really get it but okay
All the non direct repeats gets deleted
27
When might we want to restrict HR?
When it occurs between non sister chromatids because its between non homologous strands which can lead to large deletions.
28
How can we detect whether or not there has been crossovers between sister chromatids?
- Incorporate BrdU into 1 strand during DNA replication = this allows us to see crossovers between sister chromatids - We can add DNA damaging agents then do the same to see the cross overs = leads to imbalanced translocations
29
Why is HR a double edged sword?
- Can lead to loss of heterozygosity which may be needed. | - Occurs between non allelic sequences by ectopic recombination (deletions/translocations/inversion)
30
How does loss of heterozygosity occur?
Crossing over between homologues instead of sister chromatids, leads to daughter cells that are purely dominant or recessive, can make it cancerous (escalated fast idk)
31
When is HR with homologue beneficial and why?
During meiosis to promote genetic variation in gametes.
32
What happens if homologue recombination doesn't occur during meiosis?
Meiosis won't be completed due random chromosome reductional segregation lol k
33
What are the phentoypes of cell lines deficient in HR?
- Genomic instability even without DNA damaging agents because there will be bare replication stalling - Not that sensitive to IR (DSBs caused by IR usually repaired by NHEJ) - Mildly sensitive to UV = DSB - Really sensitive to DNA cross linking agents - Really sensitive to agents that block SSBR (parp inhibitors)
34
What are the disorders associated with defects in HR?
- Breast cancer - BLM and WRN - ATLD and Nbs1
35
How is Brca1 involved in DSB repair?
End resection via interaction with CTIP
36
How is Brca2 involved in DSB repair?
Direct mediator of RAD51 and also displaces RPA from ssDNA
37
Why does defect in HR lead to breast cancer?
Because Brca1 and 2 are involved in DSB repair so cells now sensitive to DSB. Also, patients may suffer loss of heterozygous event?
38
Describe Bloom (BLM)
It is a rare autosomal recessive disorder characterised by short stature, genomic instability due to excessive HR, progeria like cancer predisposition. - Mutation in BLM helicase promotes resolution COs = loss of heterzygosity and gross chromosomal rearrangments
39
Describe Werner (WRN)
- Similar to BLM - Premature ageing - Mutation in helicase promotes resolution COs = loss of heterzygosity and gross chromosomal rearrangements
40
Describe ATLD and Nbs1
- It is a rare autosomal recessive congenital disorder causing chromosomal instability, leading to microcephaly, short stature, immunodeficiency, radiation sensitivity, predisposition to lymphoid malignancy (THIS WASN'T ON THE SLIDE, I ADDED IT FOR BETTER UNDERSTANDING) - MRN deficient - MRN required in HR so if its defective it would lead to problems with DSB repair

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