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Genetics Gary > Lecture 3- recombination > Flashcards

Lecture 3- recombination Flashcards

1
Q

DNA recombination

A

The means by which organisms can shuffle their genes.

Recombination deals with DNA double stranded breaks

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2
Q

ssDNA

A

Single stranded DNA

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3
Q

dsDNA

A

Double stranded DNA

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4
Q

Duplex

A

Double stranded DNA

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5
Q

Recombination

A

Exchange of genetic material

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6
Q

Crossover/splice/recombinant

A

Recombindation where flanking markers are exchanged

non-crossover/patch/non-recombinants:recombination where flanking markers are not exchanged

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7
Q

Homoduplex

A

A DNA molecule composed of two strands with each derived from the same parent chromosome

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8
Q

Heteroduplex

A

DNA molecule composed of two chains with each derived from a different parent chromosome

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9
Q

Gene conversion

A

A mismatched DNA sequence from one heteroduplex DNA strand is replaced with a sequence complementary to the other strand resulting in aberrent gamete ratios in meiosis

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10
Q

Spontaneous DNA damage

A

Degradation of DNA (loss of bases or amine groups from bases)
Metabolic products (reactive oxygen species)
Misincorporation: of bases or errors by DNA polymerases (mismatches) and DNA repair

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11
Q

Induced DNA damage

A

Mis-repair of damage caused by radiation 9UV light, X-rays)

Methylation of bases (crosslinking reagents)

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12
Q

Homologous recombination

A

Repair of a double strand DNA break.
Partner chromosomes are found and single strands exchanged to form DNA branched structures.
Exchanges provide a 3’ end to prime replication restart or offer a template to copy any genetic information that may be lost.

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13
Q

How does homologous recombination conserve genetic identity?

A

Aligns chromosomes at meiosis- chiasma form to link chromosomes so they segregate appropriately into separate cells
Repairs DNA breaks ‘Restores stalled replication forks

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14
Q

How does homologous recombination generate genetic diversity?

A

Re-arranges genes within a genome
Exchanges homologous partners
Incorporates foreign DNA
Contributes to acquisition of new traits

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15
Q

What is the basis of homologous recombination?

A

Partner chromosomes are found and single strands are exchanged to form DNA branched structures. Exchanges provide a 3’ end to prime replication restart or offer a template to copy and genetic information that may be lost.

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16
Q

Model of homologous recombination

A

Robin Holliday proposed a model to provide an explanation of recombinants that he observed in different fungi.

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17
Q

What are the two possible products of homologous recombination?

A

2 non-recombinant chromosomes with short heteroduplex regions
OR
2 recombinant chromosomes with short heteroduplex regions

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18
Q

Describe the evolution of Holliday junction models

A

Meselson & Radding proposed that a nick in one chromosome primes DNA synthesis (from the 3’ end) that displaces a strand to form an invasive ssDNA tail that ultimately yields a Holliday junction.
Szostak proposed a model where recombination is initiated at a double strand DNA break (DSB) and the formation of two Holliday junctions. DsDNA break then the ends are exchanged to form a Holliday junction that expands to form 2.

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19
Q

Describe the Messelson-Radding model of the Holliday junction
(Nick initiated model)

A

Endonuclease makes a cut
DNA synthesis occurs displacing the nicked strand
The displaced strand invades the second chromatin
The remaining free ends are ligated forming a Holliday junction
Only one of the molecules has a heteroduplex region which results in asymettry. The junction may move to create two heteroduplexes.
The junction is resolved with a horizontal or vertical cut.

20
Q

Repairing a dsDNA break by homologous recombination mediated pathways

A

Double strand break repair (DSBR)

Synthesis dependent strand annealing (SDSA)

21
Q

What do both repair pathways of dsDNA break by homologous recombination have in common?

A

In both pathways repair is initiated by resection (degradation of one strand) of a DSB to provide 3’ single stranded DNA overhangs

22
Q

Homology dependent double strand break repair

A

Parental DNA duplex broken down by a double stranded break. Nuclease degradation occurs.
Recombination proteins allow the 3’ ended molecule to invade the homologous chromosome molecule at a region of homology to form a heteroduplex region.
The displaced strand forms a loop (D loop)
The 3’ end of the invading strand acts as a primer for DNA synthesis.
The bubble closes once DNA replication is complete.

23
Q

Recombination can be split into SYNAPTIC stages. What are these?

A

Pre-synapsis
Synapsis
Post-synapsis

24
Q

Pre-synapsis (recombination)

A

Events take place that are needed for the initiation of recombination. This stage includes the formation of faps, single or double strand breaks and the exposure of single stranded regions

25
Q

Synapsis (Recombination)

A

Homologous strands are paired up and strand exchange takes place to produce 3 then 4 stranded branched DNA intermediates

26
Q

Post-synapsis (Recombination)

A

the 4 stranded Holliday junctions are cut to give cross over or non-cross over products

27
Q

1 Branch migration
2 Resolution
3 Mismatch repair

A

1 moves the branch along the DNA
2 involves strand cleavage at the Holliday junction to separate the linked chromosomes
3 Corrects and base pair mistakes

28
Q

RecA function

A

Homologous pairing and strand exchange

29
Q

RecB,C,D function

A

Helicase-exonuclease at ends, helps load RecA on 3’ overhangs

30
Q

Rec F,O,R function

A

Helps load RecA at single strand gaps in DNA

31
Q

RecG

A

Holliday junction and branch migration helicase

32
Q

RecJ

A

5’-3’ single strand DNA exonuclease, trims end

33
Q

SbcB

A

3’-5’ single strand DNA exonuclease, trims end

34
Q

RuvA,B,C

A

Holliday junction branch migration and resolution

35
Q

RecQ

A

DNA helicase, unwinds DNA secondary structures and junctions

36
Q

What is RecBCD?

A

An exonuclease that only works at the double stranded ends- will cut away one of the ends

37
Q

RecBCD - Rec B and Rec D

A

helicases that travel along each of the strands at the DNA break
Bipolar helicases as they unravel DNA but move in opposite directions. Rec B moves in 3’ to 5’ and RecD moves in 5’ to 3’

38
Q

In RecBCD how are strands nicked?

A

RecB has a nuclease domain that cuts both DNA strands

The 3’ end strand is cut more regularly and the 5’ end is cut occasionally

39
Q

What happens when RecBCD reaches a chi site?

A

Cutting of the 3’ strand ceases, but accelerates on the 5’ strand. This creates a single stranded tail.
RecA loading is directed onto the 3’ end
RecC tightly binds to the Chi site

40
Q

Pre-chi

A

Strands are separated and passed through the complex. The DNA loops as RecD works faster than RecB.

41
Q

Post-Chi

A

RecA loaded onto the 3’ strand

42
Q

Describe RecA

A

A small protein that binds ssDNA
RecA polymerises on the ssDNA to form a helical nucleoprotein filament that can be seen by electron microscopy.
RecA catalyses the homologous DNA pairing and strand exchange stages of recombination.
RecA initiates a search for a homologous duplex

43
Q

RecA and homologous pairing

A

The single strand lies through the centre of the RecA filament.
Homologous pairing is a random process and is followed by strand exchange
Strand exchange

44
Q

Strand exchange and a triple helix

A

Initially the ssDNA guanine interacts with the G-C base pair of the incoming dsDNA.
The C rotates around its phosphodiester backbone. An intermediate forms where all three bases interact with each other. The C forms a new pairing with the ssDNA G while the dsDNA G is displaced.

45
Q

Link RecA to dsDNA breaks

A

A double stranded DNA break arises by exposure to ionising radiation. The ends are processed by RecBCD exonuclease to generate 3’ ssDNA overhangs.
RecA is loaded to initiate a search for an undamaged homologous chromosome.
RecA mediates homologous pairing and strand exchange to form a D loop that is extended into Holliday junctions

Genetics Gary (9 decks)

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