Genetic Recombination Flashcards
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
What do DSBs initiate?
Recombination
Initiated by endonuclease which cleaves the recipient strand
Spo11 in meiosis
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)
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
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
What happens when the 2 Holliday Junctions are resolved…
(1) In OPPOSITE ways?
(Strands nicked at recombinant joint)
A CROSSOVER is produced
What is Branch Migration?
The migration of a Recombinant Joint along the DNA Duplex
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
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
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
NHEJ is normally fine in somatic cells. But how can it cause issues?
Homologous recombination can occur between TRANSPOSABLE ELEMENT regions
What is Site-Specific Recombination?
Recombination between DEFINED sequences - mediate VERY SPECIFIC rearrangements
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
What enzymes catalyse Site-Specific Recombination?
Tyrosine + Serine RECOMBINASES
What is a real-world example of Site-Specific Recombination?
- Integration & Excision?
Phage LAMBDA Lysogeny
Integrates & Excises from bacterial genome
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
What happens during Chromosome Dimer Resolution in Bacteria?
XerCD recombinase
MEDIATES re-recombination between dif genes
This resolves chromosome dimers, which CAN’T segregate
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)
( ! ) What FOUR mechanisms of recombinations did we study in this course?
- Homologous Recombination
- NHEJ
- Site-Specific Recombination
- Transposition
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?
- Heteroduplex DNA (formed from aligned ssDNA break)
ss break between B + C leads to recomb between A + B
Appears like double recombinant
- Heteroduplex DNA + Mismatch Correction (leads to non-reciprocality)
What can MISMATCH CORRECTION following heteroduplex formation lead to?
Negative interference
Non-reciprocality
