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Molecular Biology of the Gene > Recombination > Flashcards

Recombination Flashcards

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1
Q

What type of DNA damage is homologous recombination used to repair predominantly?

A

Double-strand breaks.

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2
Q

What is recombination?

A

Recombination is any exchange between (or itegration of) one DNA molecule and another.
Recombination isn’t defined by the mechanism by which it occurs but it is defined by the physical fact that a piece of DNA from one molecule has been incorporated into another strand.

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3
Q

What are the 3 basic types of recombination?

A
  1. Homologous recombination.
  2. Non-homologous recombination.
  3. Site-specific recombination.
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4
Q

What must happen for DNA damage to become a mutation?

A

It must be inherited.

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5
Q

What is homologous recombination?

A

The exchange of a single strand of DNA from one DNA duplex to a second DNA duplex with very similar or identical sequences.

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6
Q

What are the 2 common situations in which homologous recombination is employed?

A

DNA damage repair.
Meiosis.
*Also in mating type switching in yeast.

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7
Q

Why does homologous recombination occur in meiosis?

A

To facilitate genetic exchange and ensure accurate pairing and thus segregation of homologous chromosomes.

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8
Q

What is the basic mechanism of homologous recombination?

A
  1. Double strand DNA break occurs.
  2. The cell chews away a piece of DNA making a bigger hole.
  3. The ends of the DNA search for a homologous partner.
  4. They find some sequence they can use to copy themselves.
  5. They invade it and kick out a strand and takes its place.
  6. New DNA sythesis happens at both ends.
  7. This generates a double holliday junction (where the pieces of DNA are crossing over)
  8. There is branch migration to take the holliday junction away from the site of damage.
  9. DNA ligation fixes the nicks left over.
  10. Holliday junctions are resolved.
  11. Resolution can result in crossing over or not.
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9
Q

Why can’t homologous recombination occur prior to DNA replication?

A

Because there isn’t a homologue present and so homologous recombination cannot be used for repair.

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10
Q

Is the sister chromatid or the corresponding chromatid on the homologous chromosome more commonly used for repair?

A

The sister chromatid, purely because it is closer.

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11
Q

Why and how is the 3’ overhang produced in homologous recombination?

A

An exonuclease degrades the 5’ ends of the cut DNA to produce 3’ single stranded DNA overhangs.
This occurs because DNA polymerase can only add to the 3’ end anad the 3’ overhang is now sufficient to search for homologous chromosomes.

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12
Q

How much homology is needed for homologous recombination?

A

15bp.

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13
Q

What is strand invasion?

A

One of the 3’ overhangs pairs with the complementary strand on the homologous DNA molecule and displaces the strand (via triple-stranded intermediate) to create a D loop.

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14
Q

What is the D loop?

A

The D loop is the region of displaced DNA that remains in tact, the displaced DNA in the D loop is never broken.

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15
Q

Why does branch migration occur?

A

Branch migration occurs to take the crossover point away from where the original damage was.
This takes the crossover formed by the holliday junction away from the site of initial damage.

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16
Q

How many more recombination events are there in meiosis than there are crossover events in meiosis?

A

9x

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17
Q

How do we get crossover during recombination?

A

When the 2 different mechanisms are used to resolve each holliday junction.

18
Q

In what situation can crossovers occur?

A

Meiotic crossing over between homologous chromosomes.

Sister chromatid exchanges.

19
Q

What do non-crossover event lead to?

A

Gene conversion.

20
Q

In what organims did homologous recombination first evolve?

A

Prokaryotes - bacteria.

21
Q

Why don’t we study meiotic recombination in E.coli?

A

Prokaryotes don’t do meiosis, so we use S. cerevisiae instead.

22
Q

What generates double strand breaks?

A

DNA damage e.g. directly by irradiation.
HO endonuclease makes double strand breaks in mating type loci in yeast.
Spo11 is an endonuclease that cuts DNA initiating double strand repair pathway in meiosis to allow accurate chromosome pairing and segregation through mieotic division and to allow genetic variation.

23
Q

What processes the broken ends of a double-strand break to generate ssDNA for strand invasion?

A

A 3-protein complex RecBCD helicase/nuclease.

24
Q

What are RecB and RecD?

A

Helicases (RecB also a nuclease) which digest DNA and use ATP to unwind DNA to generate 3’ overhang and recognise a site on the DNA that says stop.

25
Q

What is RecA?

A

A single stranded DNA binding protein.

26
Q

What is the function of RecA?

A

Cells don’t like having naked DNA in the cell so the 3’ overhang is coated with ssDNA binding protein RecA.
RecA loads as monomers onto the ssDNA forming a nucleo-protein filament that stretches out the DNA exposing the bases on the DNA facilitating homology searching and promoting strand invasion.
RecA can bind more than one DNA molecule so promotes interaction between different strands.

27
Q

How does homology searching occur?

A

Bases are flipped out of the dsDNA helix and they interact with the ssDNA.

28
Q

What is a holliday junction?

A

After initial strand exchange, the two DNA molecules are connected at a holliday junction.

29
Q

Which molecule promotes continued strand excahnge to move the holliday junction?

A

RuvA

30
Q

What is the function of RuvA?

A

RuvA promotes continued strand exchange allowing movement of the holliday junction.
RuvA is the strucutral component holding the junction in place in an unfolded planar configuration.
There are 2 RuvA tetramers per holliday junction.

31
Q

How does RuvA recognise the holliday junction?

A

By structure (not by sequence)

32
Q

What is RuvB?

A

RuvB is a hexameric ATPase driving exchange of base pairs.

33
Q

What is the function of RuvB?

A

There are 2 RuvB helicases per holliday junction pulling DNA from the top and thebottom and hence they work in opposite directions to drive migration.
RuvB hexamers work along the molecule pushing the molecule out one way and pulling it in from the other side promoting branch migration.

34
Q

What terminates branch migration?

A

RuvC evicts RuvB which would continue promoting branch migration forever if it weren’t for RuvC.

35
Q

What is the function of RuvC?

A

RuvC recognises the holliday junction with RuvA and RuvB and evicts RuvA and goes in its place and slows down the rate of branch migration acting as a brake.
RuvC also cleaves the DNA to resolve the junction, RuvC is the one that makes the decision on how to resolve the junction/how to cleave.

36
Q

How does RuvC recognise the holliday junction with RuvA and RuvB still attached?

A

Structure (not sequence).

37
Q

What is the identity of the resolvase in eukaryotes?

A

Mus81/Mms4 (although still disputed).

38
Q

What kind of recombination is site-specific recombination?

A

Non-homologous recombination.

39
Q

Give 3 examples of where site-specific recombination occurs?

A

Bacteriophage insertion.
V(D)J recobination (aquired/adaptive immunity).
Plasmid structural rearrangement.
*All catalysed by recombinases.

40
Q

Give 3 examples of recombinase systems?

A
  1. Lambda integrase; att sites (from Bacteriophage integration in E. coli).
  2. Cre recombinase; LoxP sites (from Phage P1).
  3. Flp recombinase; FRT sites (from S. cerevisiae 2 micron plasmid).

Molecular Biology of the Gene (5 decks)

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