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Flashcards in DSB 3 Deck (40)
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1

What is the main difference between HR and NHEJ?

HR requires a repair template

2

When does HR occur?

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

3

When are sister chromatids not used in HR?

During meiosis

4

Describe the general HR pathway

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

5

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

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

6

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

- Double Holiday Junction model (DSBR)

- Synthesis dependent strand annealing (SDSA)

- Break induced replication (BIR)

- Single strand annealing (SSA)

7

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

Because SSA can lead to large deletions

8

In what direction does DNA resection occur?

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

9

Why is the 3' end so important?

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

10

Describe the HR pathway is detail

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

11

Why is it dangerous to expose lots of ssDNA?

- 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

12

Why does HR use the sister chromatid?

Because it is super close and it is identical obvs

13

What is heteroduplex DNA?

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

14

Why is the holiday junction a thermodynamic structure?

- 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

15

When do heteroduplex DNAs arise?

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

16

What happens as a result of heteroduplex DNA?

Either mismatch repair or inherited as mutation

17

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

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

18

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

Full: the full duplex is intertwined

Hemi: 2 of the ss from separate molecule is intertwined

19

What is the difference between crossovers and noncrossovers?

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

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

20

When is crossover essential and repressed?

- Essential in meiosis

- Repressed in somatic cells

21

What is the difference between dissolution and resolution?

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

What are the classic resolvases?

- Gen 1 in mammalian cells

- Yen1 in yeast

- Othologues of RuvC (classic bacterial resolvase)

23

What is synthesis dependent strand annealing (SDSA)?

Another repair process that requires HR

- 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

What is break induced replication? BIR

Another process for repair that requires HR

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)