1

Recombination
 2
Group Members

 Muhammad Khaleeq Saqi (FA22-RBM-005)

 Mehran Ahmad Khan (FA21-RMV-003)

 Syed Muhammad Omer (FA21-RMV-010)

 Sabahat Ali (FA22-RBM-007)

 Seemab Zahra (FA22-RBM-009)

 Beenish Idrees (FA22-RBM-002)

12/31/2022
 3

Objectives

 What is Recombination
 Types of recombination
 Models of recombination
 Recombination in Prokaryotes and Eukaryotes
 Factors involved in Prokaryotic and Eukaryotic
Recombination
 Chromatin Context in Recombination
12/31/2022
 4
Recombination

 The process in which the pieces of DNA are broken and recombine
to form new combinations of allele

 It creates genetic diversity at gene level that reflect differences in

DNA sequences of different organisms

 In eukaryotes recombination typically occur during meiosis

12/31/2022
 5
Crossing over

 Crossing over is the exchange of genetic material during

sexual reproduction between two homologous chromosomes'
non-sister chromatids that results in recombinant
chromosomes

 Crossing over takes place during meiosis

 During alignment, arms of chromosomes overlap temporarily

fuse and cause crossing over
12/31/2022
 6
Stages of Meiosis

 Meiosis I Leptotene
Chromosome
 Meiosis II Diakinesis
condense
Attach to
nuclear Zygotene
Meiosis I: Chromosome
condensaton
membrane Synapsis
Synaptonemal
Nuclear complex
 Prophase I membrane
disintegrates

Metaphase I Diplotene
Synapsis ends
Pchytene
Crossing over
Homologous between non-
Anaphase I pair remain
attach at
sister
chromatids
chaismata

Telophase I Sub-stages of prophase

I 12/31/2022
 7
Sub-stages of prophase I

12/31/2022
 8
TYPES OF RECOMBINATION

Homologous recombination
Non-Homologous recombination
Site specific recombination
Replicative recombination
12/31/2022
 9
1. HOMOLOGOUS RECOMBINATION

 Nucleotide sequences are exchanged between two similar or

identical molecules of DNA.

12/31/2022
 10
2. Non-homologous Recombination

 Refers to any DNA rearrangement

that leads to covalent joining of non
homologous linear DNA segments

 Occurs in most of gram positive

bacteria

12/31/2022
 11
3. SITE SPECIFIC RECOMBINATION

 Occurs between particular

short sequences

 It requires a special
enzymatic machinery for
each particular site.

12/31/2022
 12
4.REPLICATIVE RECOMBINATION

 Which generates a new

copy of segment of DNA

 Many transposable
elements use the
process of replicative
recombination

12/31/2022
 13

Model of recombination

 Holliday Model

 Meselson-Radding Model

 Double Stranded Break Repair Model

12/31/2022
 14

Holiday Model:

 Proposed by Molecular Biologist, Robin

Holliday in 1964

 a model of meiotic recombination in Holliday junction from X ray crystallography

which homologous chromatids of a RuvA-Holliday junction complex.
exchange single DNA strands to form
a joint molecule with a four-way
junction at the point of exchange.

12/31/2022
Electron micrograph of a Holliday junction
 15
Steps Of Holliday Model:

 Single Stranded Nicks formed on both DNA Duplexes

 Base pairs form between the two recombining DNA duplexes
 The heteroduplex formed as the holiday junction move
 The Holliday junction is cleaved, regenerating two separate DNA duplexes
 Resolution results in
→ Crossover product in Vertical cut
→ Non-crossover Product in Horizontal cut

12/31/2022
16

12/31/2022
 17
Meselson-Radding Model

 This model was proposed by Meselson and Radding in 1975.

 A model that explains genetic combination through asymmetric

heteroduplex.

 The Endonuclease enzyme is used to introduce single strand nicks.

12/31/2022
 18
Steps Of Meselson-Radding Model

1. Single Strand Nick produced in one of DNA duplex

2. Strand Invasion
3. Branch Migration
4. Ligation of strand
5. Resolution

12/31/2022
Meselson and Radding Model
19

12/31/2022
 20
DOUBLE STRAND BREAK REPAIR MODEL:

 The model Given by S.Jack and colleagues.

 In the double-strand-break model, the region corresponding to the

original gap now has the sequence of the donor duplex in both
molecules.
 DSB could lead to chromosomal rearrangements, tumorigenesis or even
cell death.

12/31/2022
 21

Steps of DSBR Model:

 An endonuclease cleaves both strands of one of the homologous DNA duplexes.

 The cut is enlarged by an exonuclease to generate a gap.
 One of the free 3'ends invades a homologous region on the other duplex called
the donor duplex.
 The D loop is extended as a result of repair synthesis primed by the invading 3'end.
 When the displaced strand from the donor, extends as far as the other side of the gap on the
recipient, it will anneal with the other 3' single stranded end at that end of the gap. The
displaced strand has now filled the gap on the heteroduplex. DNA polymerase converts the
donor D-loop to duplex DNA.
 DNA ligase will seal the nicks, sealing the nick forms a Holliday junction.

12/31/2022
DSBR Model 22

12/31/2022
 23
Recombination in Prokaryotes

In Prokaryotes, genetic recombination occurs through the unilateral

transfer of DNA.
This includes;
Transduction
Transformation
conjugation

12/31/2022
 24

Recombination in Prokaryotes

Transduction Transformation Conjugation

• Gene transfer is • Process of horizontal • Bacterial conjugation is the
mediated by viruses gene transfer transfer of genetic material
• Bacteriophages from donor bacterium of a
attack bacteria and • Bacteria take up
mating pair into recepient.
carry genes from foreign genetic
one bacterium to material (naked • Direct cell contact or by a
another DNA) from the bridge like connection
environment. called F-pilus

12/31/2022
 25
Recombination in Prokaryotes

12/31/2022
 26

Recombination In Eukaryotes
Alternative double-strand break repair (DSBR)
mechanisms.
(NHEJ) or (Alt-NHEJ) (Error prone mechanisms)
occurs by
1) direct ligation of the broken DNA strand
2) ligation after minimal processing.
Within direct repeat sequences, DSBR could occur by single-
strand annealing (SSA).
SSA causes loss of a repeat sequence due to direct resection,
annealing, and ligation.
Synthesis-dependent strand annealing (SDSA)
Nascent strand is synthesized, displaced by D-loop
dissociation, and anneals with the other 30 ssDNA overhang
to complete DNA synthesis.
SDSA results in non-crossover products. D-loop can also be
cleaved to produce crossover products
12/31/2022
 27
DNA double strand break repair by
homologous recombination

 Initiated by D-loop formation (strand invasion)

 Invading DNA strand primes DNA synthesis

 In double Holliday junction (dHJ), the second-end is

captured and the strands are ligated after DNA
synthesis.

 Branch migration results in non-crossover products

or stabilize dHJs to undergo resolution.
dHJ resolution can result in either crossover or non-
crossover products.
12/31/2022
 28
Break-induced replication (BIR)

 Initiates from a single-ended strand invasion.

If one arm of the chromatid is lost after the
double-strand break (DSB OR if a telomere is
uncapped, a 30overhangisformed.

 DNA synthesis can continue to the end of the

chromatid by migration of the D-loop after D-
loop cleavage.

12/31/2022
 29
Homologous recombination (HR) restores
collapsed or stalled replication forks.

 Nicks of template strands

 lead to replication fork collapse, which
can be repaired by:
o D-loop formation and Holliday
junction cleavage to restore
replication.
 Lesion on a leading strand
 result in replication fork regression and
DNA synthesis on the leading strand.
o Subsequent branch migration
restores the replication fork.

12/31/2022
 30
Pathways of recombination in DSB
repair.

1) pre-synapsis, (Step 1-2)

2) synapsis, (Step 3)
3) post-synapsis.

In synthesis-dependent strand annealing (SDSA, steps 4a - 5a - 6a),

disengaged, leading to localized conversion→ without crossover.
In break-induced replication (BIR, steps 4a - 5b - 6b),
D-loop assembled into a full replication fork, copying the entire distal part
of the chromosome to result in loss-of heterozygosity (LOH).

In double-strand break repair (DSBR, steps 4b - 5c - 6c-e - 7), both

ends of the DSB are engaged, either by independent strand invasion or
by second end capture, leading to double Holliday junction formation.
Resolution of junction into crossover products and non-crossover
products (steps 6c and d)
or BLM-mediated branch migration and TOPOIIIα-catalyzed dissolution
of a hemi-catenane (step 6e),
leading exclusively to non-crossover products (step 7).
12/31/2022
 31
Prokaryotic and eukaryotic factors that
catalyze recombination steps

Recombination steps E.coli protein catalyst Eukaryotic protein catalyst

Pairing Homologous DNA and Rec A protein Rad51

strand Invasion
Introduction of DSB
Processing DNA breaks
to generate single
strands for invasion
Assembly of strand-exchange
proteins
Holiday Junction recognition
and branch migration
Dmc1 (in meiosis)
None Spo11 (in meiosis)
HO for mating type switching
RecBCD helicase/nuclease MRX Protein
RecBCD and RecFOR Rad52/Rad59
RuvAB complex Not well characterized
12/31/2022
 Chromatin-mediated regulators of meiotic 32
recombination revealed by proteomics of a
recombination hotspot

 a Binding of Atf1-Pcr1 heterodimer to an M26 DNA

sequence motif promotes the catalysis of
recombination-initiating DSBs by Rec12 (Spo11).
 b Hotspot-specific, meiotically induced chromatin
remodeling, involving histone PTMs (lollipops) and
the displacement of nucleosomes (ovals),
generates access to DNA and potential docking
moieties for the basal recombination machinery and
its catalytic subunit, Rec12 (Spo11).
 c Sequences of alleles used in this study. Each
allele contains bp substitutions (bold) that create a
stop codon (italics) in the ade6 ORF. Hotspot
alleles contain an M26 DNA site (underlined) to
which the Atf1-Pcr1 heterodimer binds
Fig. Features of the ade6-M26 meiotic recombination hotspot 12/31/2022
 33
References

 1. Branzei D, Szakal B. DNA helicases in homologous recombination repair.

Current Opinion in Genetics & Development. 2021 Dec 1;71:27–33.
 2. Giaccherini C, Gaillard P. Control of structure-specific endonucleases during
homologous recombination in eukaryotes. Current Opinion in Genetics &
Development. 2021 Dec 1;71:195–205.
 3. Huselid E, Bunting SF. The Regulation of Homologous Recombination by
Helicases. Genes. 2020 May;11(5):498.
 4. Verma P, Greenberg RA. Communication between chromatin and homologous
recombination. Current Opinion in Genetics & Development. 2021 Dec 1;71:1–9.
 5. Young SJ, Sebald M, Shah Punatar R, Larin M, Masino L, Rodrigo-Brenni MC,
et al. MutSβ Stimulates Holliday Junction Resolution by the SMX Complex. Cell
Reports. 2022 Oct 20;33(3):108289.

12/31/2022