Homologous recombination

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SUBMITTED TO: SUBMITTED BY:
Miss Gitanjali Ankush Yadav
Botany department ROLL NO. : 25

INTRODUCTION
• Recombination is the term generally used to
describe outcome of crossing-over between pairs
of homologous chromosomes during meiosis.
• In the 1960s , models were proposed for the
molecular events that underlie crossing-over ,
and it was realized that a key part of molecular
recombination is the breakage and subsequent
rejoining of DNA molecules .
• Biologists now use “recombination” to prefer a
variety of processes that involves the breakage
and union of polynucleotides. These include :

• Homologous recombination, also called general
recombination, which occurs between segments of DNA
molecules that share extensive homology. These segment
might be present on different chromosomes, or might be
two parts of a single chromosome. Homologous
chromosome is responsible for crossing-over during
meiosis.
• Site specific recombination, which occurs between DNA
molecules that have only a short region of sequence
similarity, possibly a few base pairs . Site specific
recombination is responsible for the insertion of page
genome into bacterial genome .
• Transposition, which result in transfer of a segment of DNA
from one position in the genome to another.

Reference: Molecular biology of the gene (seventh addition) 2012

MODELS FOR HOMOLOGOUS RECOMBINATION
• THE HOLIDAY MODEL :
1. Alignment of two homologous DNA molecules. By
homologous we mean that the DNA sequences are
identical or nearly identical for a reason of at least 100
base pairs or so. Despite this high degree of similarity,
DNA molecules can have small region of sequence
difference and, may for example, carry different
sequence variants known as alleles, of the same gene.

2. Introduction of breaks into the DNA. The break occur in one DNA
strand or involve both DNA strands.
3. Formation of initial short regions of base pairing between the two
recombinant molecules. This pairing occur when a single stranded
region of DNA originating from one parental molecule pairs with its
complementary stand in homologous duplex duplex DNA molecule.
This step is known as strand invasion . As a result of strand invasion,
the two DNA molecules become connected by crossing DNA strands.
This structure is called a Holliday Junction .

5. Cleavage of holiday junction. Cutting the DNA strands within the
holiday junction regenerates two separate duplex DNA
molecules, and therefore finishes genetic exchange. This
process is called resolution.
6. If the two DNA molecules are not identical but, for example,
carry a few small sequence differences, as is true often between
two alleles of the same gene- branch migration through these
regions of sequence difference generates DNA duplex carrying
one or a few sequence mismatches (see B and b alleles) . Such
regions are called heteroduplex DNA .

Holiday junction cleavage
Reference: Molecular
biology of the gene
(seventh addition) 2012

THE DOUBLE-STRAND BREAK REPAIR MODEL
• The initiating event is the
introduction of a double
strand break (DSB) in one
of the two DNA
molecules.
• After introduction of the
DSB, a DNA-cleaving
enzyme sequentially
degrades the broken DNA
molecule to generate
regions of single-stranded
DNA (ssDNA).
• This processing creates
single-strand extensions,
known as ssDNA tails, on
the broken DNA
molecules; these ssDNA
tails terminate with 30
ends.

• The ssDNA tails generated
by this process then invade
the unbroken homologous
DNA duplex.
• The invading strand base-
pairs with its
complementary strand in
the other DNA molecule.
• Because the invading
strands end with 3′ termini,
they can serve as primers
for new DNA synthesis.
Elongation from these DNA
ends—using the
complementary strand in
the homologous duplex as a
template serves to
regenerate the regions of
DNA that were destroyed
during the processing of the
strands at the break site.

• If the two original DNA duplexes were not
identical in sequence near the site of the break
(e.g., having single-base-pair changes as
described above), sequence information could be
lost during recombination by the DSB-repair
pathway.
• The two Holliday junctions found in the
recombination intermediates generated by this
model move by branch migration and ultimately
are resolved to finish recombination

HOMOLOGOUS RECOMBINATION PROTEIN MACHINES
• Organisms from all branches of life encode
enzymes that catalyze the biochemical steps of
recombination.
• In some cases, members of homologous
protein families provide the same function in all
organisms. In contrast, other recombination
steps are catalyzed by different classes of
proteins in different organisms but with the
same general outcome

Prokaryotic and eukaryotic factors that catalyze
recombination steps :
Recobination step E.coli protein catalyst Eukaryotic Protein catalyst
Pairing homologous DNAs
and strand invasion
RecA protein Rad51
Dcm 1 (in meiosis )
Introduction of DSB None Spo11 (in meiosis)
HO ( for mating type
switching)
Processing of DNA breaks
to generate single strands
of invasion
RecBCD
Helicase/nuclease
MRX Protein (also called
Rad50/58/60 nucleases )
Assembly of strand
exchange protein
RecBCD and RecFOR Rad52 and Rad59
Holiday junction
recognition and branch
migration
RuvAB complex unknown
Resolution of holiday
junction
Ruvc Perhaps Mus81 and others

The RecBCD Helicase/Nuclease Processes Broken DNA
Molecules for Recombination
• The RecBCD enzyme processes broken DNA molecules to generate
these regions of ssDNA.
• RecBCD is composed of three subunits (the products of the recB, recC,
and recD genes) and has both DNA helicase and nuclease activities.
• The activities of RecBCD are controlled by specific DNA sequence
elements known as Chi sites.
• The RecB and RecD subunits are both DNA helicases, that is, enzymes
that use ATP hydrolysis to melt and unwind DNA base pairs.
• RecB subunit contains a 3' to 5' helicases and has also a multifunctional
Nuclease domain that digests the DNA as it moves along. RecD is a 5' to
3' helicase, and RecC functions to recognize Chi sites.

• The nuclease activity of RecBCD frequently cleave each strand during unwinding and thereby
destroys the DNA.
• Upon encountering the chi sequences the nuclease activity of the RecBCD enzyme is altered.
• After encounter with chi site, the other DNA strand (the one with 5' to 3' polarity) is cleaved
even more frequently then it was prior to chi site.
• As a result of this change in activity, the DNA duplex now has a 3' single-strand extension
terminating with the Chi sequence at the 3' end.

• The ssDNA tail generated by RecBCD must be coated by the RecA protein
for recombination to occur.
• To ensure that RecA, rather than SSB, binds these ssDNA tails, RecBCD
interacts directly with RecA and promotes its assembly.
• RecA Protein Assembles on Single-Stranded DNA and Promotes Strand
Invasion
• These proteins catalyze the pairing of homologous DNA molecules.
• Pairing involves both the search for sequence matches between two
molecules and the generation of regions of base pairing between these
molecules.
• The active form of RecA is a protein–DNA filament .
• The filament grows by the addition of RecA subunits in the 5' to 3'
direction, such that a DNA strand that terminates in 3 ends is most likely
to be coated by RecA.

• RecA–ssDNA complex is the active form that participates in
the search for a homology.
• This homology search is promoted by RecA because the
filament structure has two distinct DNA-binding sites: a
primary site (bound by the first DNA molecule) and a
secondary site
• This secondary DNA binding site can be occupied by double-
stranded.
• A sequence match of just 15 bp provides a sufficient signal to
the RecA filament that a match has been found and thereby
triggers strand exchange.
• Once a region of base-pair complementarity is located, RecA
promotes the formation of a stable complex between these
two DNA molecules.
• This RecA-bound three-stranded structure is called a joint
molecule and usually contains several hundred base pairs of
hybrid DNA.
• Strand exchange thus requires the breaking of one set of base
pairs and the formation of a new set of identical base pairs.

Model of two steps in the search for homology and DNA strand
exchange within the RecA filament

• The displacement reaction can occur between DNA molecules in
several configurations and has three general conditions:
• One of the DNA molecules must have a single-stranded region.
• One of the molecules must have a free 3′ end.
• The single-stranded region and the 3′ end must be located within a
region that is complementary between the molecules.
RecA promotes the assimilation of invading single
strands into duplex DNA as long as one of the reacting strands has
a free end.
Refrenece: lewins Genes XII 2018

• We can divide the reaction
that RecA catalyzes between
single stranded and duplex
DNA into three phases:
1. A slow presynaptic phase
in which RecA polymerizes
on single stranded DNA.
2. A fast pairing reaction
between the single-
stranded DNA and its
complement in the duplex
to produce a heteroduplex
joint.
3. A slow displacement of
one strand from the
duplex to produce a long
region of heteroduplex
DNA.

• When a single-stranded
molecule reacts with a
duplex DNA, the duplex
molecule becomes
unwound in the region of
the recombinant joint.
• The initial region of
heteroduplex DNA may not
even lie in the conventional
double-helical form, but
could consist of the two
strands associated side by
side. A region of this type is
called a paranemic joint, as
compared with the classical
intertwined plectonemic
relationship of strands in a
double helix.

Holiday junction must be resolved
• The proteins involved in stabilizing and resolving Holliday junctions have
been identified as the products of the ruv genes in E. coli.
• RuvA recognizes the structure of the Holliday junction.
• RuvA binds to all four strands of DNA at the crossover point and forms two
tetramers that sandwich the DNA.
• RuvB is a hexameric helicase with an ATPase activity that provides the
motor for branch migration.
• Hexameric rings of RuvB bind around each duplex of DNA upstream of the
crossover point.

• RuvAB displaces RecA
from DNA during its
action.
• The third gene, ruvC,
encodes an endonuclease
that specifically recognizes
Holliday junctions.
• It can cleave the junctions
in vitro to resolve
recombination
intermediates. A common
tetranucleotide sequence
provides a hotspot for
RuvC to resolve the
Holliday junction. Refrenece: lewins Genes XII 2018

Eukaryotic Genes Involved in Homologous Recombination
1 End Processing/Presynapsis
• In mitotic cells, DSBs are produced by exogenous sources such as irradiation or
chemical treatment and from endogenous sources such as topoisomerases and
nicks on the template strand. During replication nicks are converted to DSBs.
• The ends of these breaks are processed by exonucleolytic degradation to have
single-strand tails with 3′–OH ends.
• The first step in end processing entails binding of the broken end by the MRN or
MRX complex, in association with the endonuclease Sae2 (CtIP in mammalian
cells).
• MRX – Mre11, Rad50, Xrs2 (Yeast)
• MRN – Mre11, Rad50, Nbs1 (Mammalian)
• Rad50 is thought to help hold DSB ends together via dimers connected at the tips
by a hook structure that becomes active in the presence of zinc ion.
• After MRN/MRX and CtIP/Sae2 have prepared the DSB ends and removed any
attached proteins or adduct that would inhibit end resection, the ends are resected
by nucleases that act in concert with DNA helicases that unwind the duplex to
expose single-strand DNA ends.
• After the DSBs have been processed to have 3′–OH single-strand tails, the single-
strand DNA is bound first by the single-strand DNAbinding protein RPA to remove
any secondary structure.
• With the aid of mediator proteins that help Rad51 displace RPA and bind the single-
strand DNA, Rad51 forms a nucleofilament.
• Rad51 is required for all homologous recombination processes except single-strand
annealing.

Rad50 has a coiled coil
domain similar to SMC (structural
maintenance of chromosomes)
proteins. The globular end contains
two ATP-binding and hydrolysis
regions (a and b) and forms a complex
with Mre11 and Nbs1 (N) or
Xrs2 (X). The other end of the coil
binds a zinc cation and forms a
dimer with another MRX/N molecule.
The globular end binds to
chromatin. The complex binds to
double-strand breaks and can
bring them together in a reaction
involving two ends and one
MRN/X complex (top right figure) or
through an interaction between
two MRX/N dimers (bottom right
figure).
Structure for Rad50 and model for the MRX/N complex

The “head” region of Rad50, bound to Mre11, binds
DNA, while the extensive coiled coil region of Rad50 ends with a
“zinc hook” that mediates interaction with another MRN complex.
The precise position of Nbs1 within the complex is unknown, but it
interacts directly with Mre11.
MRN/X Complex

2. Synapsis :
• Once the Rad51 filament has formed on single-strand DNA in the DBSR and
SDSA processes, a search for homology with another DNA molecule begins
and, once found, strand invasion to form a D-loop occurs.
• Strand invasion requires the Rad54 protein and the related Rdh54/Tid1
protein in yeast, and RAD54B in mammalian cells.
• Although Rad54, Rdh54, and
• RAD54B are not DNA helicases, the translocase activity causes local opening
of double strands, which may serve to stimulate Dloop formation.
3. DNA Heteroduplex Extension and Branch Migration
•The proteins involved in this step are not as well defined as those required in
the early steps of homologous recombination.
•D-loop formation results in Rad51 filament being formed on doublestranded
DNA. Rad54 protein has the ability to remove Rad51 from double-stranded
DNA.
• This step might be important for DNA polymerase extension from the 3′
terminus.

4.Resolution
• The search for eukaryotic
resolvase proteins has been a
long process.
• Mutants of the DNA helicases
Sgs1 of yeast and BLM in
humans result in higher
crossover rates. These helicases
have thus been proposed to
normally prevent crossover
formation by promoting
noncrossover Holliday junction
resolution.
• The end structure is suggested
to be a hemicatenane, where
DNA strands are looped around
each other.
• This structure is then resolved
by the action of an associated
DNA topoisomerase: Top3 in
the case of Sgs1 and hTOPOIIIα
in the case of BLM. In vitro, BLM
and hTOPOIIIα can dissolve
double Holliday junctions into a
noncrossover molecule.
Double Holliday junction dissolution by the action of
a DNA helicase and topoisomerase. The two Holliday junctions are
pushed toward each other by branch migration using the DNA
helicase activity. The resulting structure is a hemicatenane where
single strands from two different DNA helices are wound around
each other. This is cut by a DNA topoisomerase, unwinding and
releasing the two DNA molecules and forming noncrossover
products.
While

Specialized Recombination Involves Specific Sites
• Specialized recombination involves a reaction between two specific sites.
• The enzymes that catalyze site-specific recombination are generally called
recombinases.
• Prominent members of the integrase family are the prototypical Int from
phage lambda, Cre from phage P1, and the yeast FLP enzyme.
 The physical condition of the DNA is different in the lysogenic and lytic
states:
1 In the lytic lifestyle, lambda DNA exists as an independent, circular
molecule in the infected bacterium.
2 In the lysogenic state, the phage DNA is an integral part of the bacterial
chromosome (called the prophage).
 Transition between these states involves site-specific recombination:
1. To enter the lysogenic condition, free lambda DNA must be inserted into
the host DNA. This is called integration.
2. To be released from lysogeny into the lytic cycle, prophage DNA must be
released from the chromosome. This is called excision.
• Integration and excision occur by recombination at specific loci on the
bacterial and phage DNAs called attachment (att) sites.

• For describing the integration/excision
reactions, the bacterial attachment site (att
) is called attB, consisting of the sequence
components BOB′. The attachment site on
the phage, attP, consists of the
components POP′.
• The sequence O is common to attB and
attP. It is called the core sequence, and the
recombination event occurs within it.
 The difference in the pairs of sites reacting
at integration and excision is reflected by a
difference in the proteins that mediate the
two reactions:
1. Integration (attB × attP) requires the
product of the phage gene int, which
encodes an integrase enzyme, and a
bacterial protein called integration host
factor (IHF).
2. Excision (attL × attR) requires the
product of phage gene xis, in addition to
Int and IHF.
• Thus, Int and IHF are required for both
reactions. Xis plays an important role in
controlling the direction; it is required for
excision, but inhibits integration.

Site-Specific Recombination Involves Breakage and Reunion
The corresponding strands on each duplex are cut at the same position, the free 3′ ends
exchange between duplexes, the branch migrates for a distance of 7 bp along the
region of homology, and then the structure is resolved by cutting the other pair of
corresponding strands.

Site specific recombination resembles tropoisomerase activity
• Integrases use a mechanism similar to that of type
topoisomerases in which a break is made in one DNA
strand at a time.
• The basic principle of the system is that four molecules
of the recombinase are required, one to cut each of the
four strands of the two duplexes that are recombining.
• The reaction involves an attack by a tyrosine on a
phosphodiester bond.
• The 3′ end of the DNA chain is linked through a
phosphodiester bond to a tyrosine in the enzyme. This
releases a free 5′–OH end.
• The free hydroxyl group of each strand then attacks the
P–Tyr link of the other strand.
• The successive interactions accomplish a conservative
strand exchange, in which there are no deletions or
additions of nucleotides at the exchange site, and there
is no need for input of energy.
• The transient 3′–phosphotyrosine link between protein
and DNA conserves the energy of the cleaved
phosphodiester bond.

Recombination Pathways Adapted for Experimental Systems
• The Cre/lox system is derived from bacteriophage P1.
• The Cre enzyme recognizes and cleaves lox sites.
• A construct is designed that is flanked by lox sites, with
the Cre gene under control of an inducible promoter
that can be turned on by temperature, hormones, or in
a tissue-specific pattern.
• Expression of Cre results in production of the Cre
protein; the Cre protein then recognizes and cleaves
the lox sites and promotes rejoining of the cut lox sites
to leave behind a single lox site, with the material
between the lox sites having been excised.

Using Cre/lox to make cell type–specific gene knockouts in mice. loxP sites are inserted into the
chromosome to flank exon 2 of the gene X. The second copy of the X gene has been knocked
out. The mouse formed with this construct is called the loxP mouse. Another mouse, called the
Cre mouse, has the cre gene inserted into the genome.

REFRENCES :
• Molecular biology of the gene (seventh addition) 2012
• lewins Genes XII 2018

Homologous recombination

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