Genetic Recombination Flashcards by Manuel Benavídez

What happens when the 2 Holliday Junctions are resolved…

(1) The SAME way?

(Same point on both strands)

No crossover

Original molecules released

They have ALTERED regions -
A footprint of the exchange

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What do DSBs initiate?

Recombination

Initiated by endonuclease which cleaves the recipient strand

Spo11 in meiosis

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What does the exonuclease (working with helicase) do in recombination?

5’ end resection

(Nibbles away one strand on either side of the DSB)

Forming two ss tails with 3’ OH ends

One nibbled end invades a homologous region in the other (donor duplex)

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How is heteroduplex DNA formed?

Exonucleases form TWO ss tails with 3’ OH ends

ONE nibbled end invades a homologous region in the other (donor) duplex

(Single-strand invasion!)

Heteroduplex formed - Base pairing between DIFFERENT parental strands

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How is a Holliday Junction formed from Heteroduplex DNA?

When Single-Strand Invasion happens, one of the donor duplex strands is displaced : D LOOP

D loop is EXTENDED by DNA synthesis (replacing degraded material)

Once it can pair with the other side, that end is captured

The second strand of the gap is filled by more SYNTHESIS and ligation

Now there are two recombinant joints - HOLLIDAY JUNCTIONS

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What happens when the 2 Holliday Junctions are resolved…

(1) In OPPOSITE ways?

(Strands nicked at recombinant joint)

A CROSSOVER is produced

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What is Branch Migration?

The migration of a Recombinant Joint along the DNA Duplex

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Which proteins recognise and resolve Holliday Junctions?

RuvA - Recognises & binds.
Its 2 tetramers sandwich the DNA.

RuvB - A helicase, catalyses branch migration

RuvC - Cleaves junctions

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How does NHEJ repair DSBs?

Enzyme detail!

DEGRADING:

Ku binds the ends

Recruits DNA-PK (a kinase)
and Artemis (a nuclease)

These degrade damaged DNA

HEALING:

Limited Microhomology lets ends come together (3-4bp)

Nucleases trim any extra

Polymerase fill any gaps

Ligase IV fills any gaps

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What is the disadvantage of NHEJ?

Information can be lost from break site - Mutagenic

Biggest problem in GERM class and G1 cells without 2 copies of a chromosome yet

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NHEJ is normally fine in somatic cells. But how can it cause issues?

Homologous recombination can occur between TRANSPOSABLE ELEMENT regions

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What is Site-Specific Recombination?

Recombination between DEFINED sequences - mediate VERY SPECIFIC rearrangements

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What THREE kinds of rearrangements can Site-Specific Recombination mediate?

INTEGRATION of a sequence

EXCISION of a sequence

INVERSION if the inserting DNA sequences sites are inverse of the genomic sequence

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What enzymes catalyse Site-Specific Recombination?

Tyrosine + Serine RECOMBINASES

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What is a real-world example of Site-Specific Recombination?

Integration & Excision?

Phage LAMBDA Lysogeny

Integrates & Excises from bacterial genome

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What is a real-world example of Site-Specific Recombination?

Inversion

(a) Flagellar phase variation in Salmonella

Direction controls expression/suppression of different flagella types (express A, repress B and then the inverse) - Escape imm System

(b) Tail fiber variation in Phage Mu
Inversion in middle of S gene - flip between two types to infect more varied hosts

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What happens during Chromosome Dimer Resolution in Bacteria?

XerCD recombinase

MEDIATES re-recombination between dif genes

This resolves chromosome dimers, which CAN’T segregate

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What 4 levels of BACTERIAL Transposon complexity did we study?

Encode TPase only
Encode drug resistance, flanked by 2 transposase sequences (1 defective)
Encode drug resistance, transposase, other genes like Resolvase (SS-Recombination system for insertion)
A bacteriophage (uses TPosition)

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( ! ) What FOUR mechanisms of recombinations did we study in this course?

Homologous Recombination
NHEJ
Site-Specific Recombination
Transposition

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What does RecA do?

Coats ssDNA - presynaptic filament

Stretches molecule by ~50%

Mediates strand exchange to form a heteroduplex

(RecA homologous is Rad51 + Dmc1 [meiosis] in eukaryotes)

Outline RecA-mediated strand exchange (3 steps)

Coated strand seeks complementarity in a ds

> Synapsis

RecA-coated strand peels off its complimentary strand and replaces it!

> Strand exchange

RecA removed (ATP step)

Which homology scenarios (placement of the non-homologous area) allowed RecA Strand Exchange?

NonHomo on 5’ end:
Synapsis AND Exchange!

NonHomo on both ends:
Synapsis only

NonHomo on 3’ end:
Synapsis only

RecA is POLAR, strand exchange only works in direction of RecA polymerisation:

5’ to 3’

How does RecA-coated DNA bind if its bases are stretched out?

The filament stretches DNA but KEEPS TRIPLETS in even spacing

Actually gives ds configuration -
there are normally 2* structures in ssDNA

What does RecBCD do?

A Helicase//Nuclease

Recognised broken ends

Unwinds strands

Digests strands (usually 3’)

Reaches Chi - switches to 5’

Then loads RecA

What do the subunits of RecBCD do??

RecB - Helicase with Nuclease RecC - Recognises Chi RecD - Helicase After Chi recognised, 3’ strand is moved out of the way and forms loop, so RecB Nuc starts cleaning the 5’ strand exclusively

What does RuvABC do?

Branch Migration & Cleave Holliday Junctions

How can we have evidence of Holliday Junctions?

Electron microscopy - slightly denaturing conditions opens junction PAGE and Ethidium Bromide - Separate by shape and weight (Rep forks are arcs, HJs are upwards spikes)

What do the subunits of RubAB do?

RuvA stretches out junction into a square planar shape Holds to facilitate branch migration RuvB - Translocase 2 rings pump DNA away from centre

What does RuvC do?

Nuclease - Actual cleavage that resolves junction and gives recombinants (crossed over or not)

What does PriA do?

Recognises junction Recruits Replicative Helicase & Machinery Synthesise missing DNA (LESS SYNTH needed in Eukaryotes) (5’ strands degraded but 3’ left)

What 2 phenotypes were used in the Meselson and Weigle (1961) Expt?

Plaque turbidity: Clear vs Turbid Plaque Size

What was the key question of the Meselson and Weigle expt?

Does recombination occur by BREAKING parental DNA?

What dsDNA markets & mutants were used in the Meselson and Weigle (1961) expt?

c I - determine plaque turbidity (mutant - clear) mi - Size of Plaque (mutant - small) There were WT (+,+) phages grown in heavy isotope ``` and mutants (c, mi) grown in normal isotopes ```

What were the RESULTS of the Meselson and Weigle (1961) expt?

Same quantity of (+, mi) recombinants as (+,+) parents Most of their DNA came from the (+,+) parent (heavy) This disproved the Copy-Choice model (Because there was heavy DNA - all new DNA is LIGHT) Parental DNA IS broken

What was the key question of the Meselson (1965) experiment?

We know parental strand is broken; Does recombination leave a SCAR in DNA?

What dsDNA markets & mutants were used in the Meselson (1965) experiment?

c I only (+) - Grown in C 13 (heavy) (c) - Grown in C 12 (light)

What were the RESULTS of the Meselson (1965) experiment?

There were MOTTLED plaques (+,c) This indicated HETERODUPLEX dna at crossover site Observed 3/4 and 3/8 heavy DNA

How do you calculate recombination frequency? | 2-Factor Cross

A: add numbers of all recombinants together B: add whole population together FR = A/B as a percentage

When should you multiply Number of Recombinants by TWO in a RF calculation?

If: 1 - Markers do not affect the RF 2 - There is RECIPROCALITY in the pop

How do you calculate the PREDICTED RF of double recombinants?

(RF of AB) x (RF of BC)

What is the Coefficient of Coincidence? (Muller, 1916)

The degree of ASSOCIATION between recombination events

What does Interference mean? | Muller, 1916

A crossover at one locus might influence the POSSIBILITY of one at a different locus

How do you calculate the CoC (S)?

OBSERVED double recombs ~ divided by ~ PREDICTED double recombs If observed = expected Random exchanges If S is <1 (more expected) POSITVE int If S is >1 (more observed) NEGATIVE int

How do you calculate interference (I) ?

I = 1-S No interference = 0 Positive if >1 Negative if <1

What was the key question of the Amati and Meselson (1965) expt?

Does Interference happen?

What were the RESULTS of the Amati and Meselson (1964) expt?

As distance between A + B decreased, CoC increased (from 1 to >1) when very close. i.e. B + C were more likely to recombine Showing NEGATIVE interference

What 2 examples can lead to negative interference?

1. Heteroduplex DNA (formed from aligned ssDNA break) ss break between B + C leads to recomb between A + B Appears like double recombinant 2. Heteroduplex DNA + Mismatch Correction (leads to non-reciprocality)

What can MISMATCH CORRECTION following heteroduplex formation lead to?

Negative interference Non-reciprocality