Lecture. 5 DNA Recombination

MOLECULAR BIOLOGY & GENETICS
Basic Processes of Molecular Biology
DNA Recombination
5th Lecture
Qurat-ul-Ain, B. Pharm., M. Phil., Ph.D.,
Visiting Assistant Professor
Department of Physical Therapy, Faculty of Health& Medical Sciences,
Hamdard University, Karachi, Pakistan
Assistant Professor
Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Hamdard
University, Karachi, Pakistan.
Qurat-ul-Ain, B. Pharm., M. Phil., Ph.D., qurat.fophu@yahoo.com

DNA Recombination
• Homologous Genetic Recombination: Involves genetic exchange between two
molecules DNA having similar sequences
• Site-specific recombination: Exchange occurs only at particular sequence on a DNA
• DNA transposition: Short segment of DNA in which chromosome moves from one
location to another

Homologous Genetic Recombination: Base-pairing between two
homologous DNA molecule
• Meiosis characteristics
1- Two Different chromosome from two
homologous DNA cross over= DNA break and
ends join to their opposite partners to re-form
two intact helices
Both of these helices contains half and half part
of both the DNA.
2- The site of cross over or the exchange of the
part of DNA molecules can occur anywhere in
the entire DNA having homologous nt
sequences in both DNA molecules
3- At the site of exchange one DNA strand make
base pairing with the other , that region called
heteroduplex joint
4- During cross over no addition or deletion of nt
takes place
This type of cross over results in DNA having
novel recombinant sequences, bc few nt
sequences are not exactly same on each strand

Homologous Genetic Recombination: Base-pairing between two
homologous DNA molecule
• This type of recombination occurs when a long region of nt sequences on both the
strands are in a match
• The point at which the cross over occur is called DNA synapsis
Qs arises that how both the strands recognize the site to start cross over ???

Homologous Genetic Recombination: Meiotic Recombination by dsDNA breaks
• DNA synapsis can only occur when anyone
of the DNA strand breaks and makes its nt
available for base pairing with the other DNA
helix
• The break in PO4 diester bond attracts the
other DNA helix to form base pairing thus
forms a synapsis
• Advance studies have shown that the
formation of synapsis starts with the help of
endonucleases that cuts both the strands at
one time
• This leaves an open 5’ and 3’ ends. 5’ ends
are chewed back by exonucleases leaving
loose 3’ end
• It is thought that these strands search base
pairing on another DNA strand having
matching or homologous sequences
• Leading to the formation of a point or joint
between maternal and paternal chromosome

Homologous Genetic Recombination: Meiotic Recombination by dsDNA breaks
Qs How the synthesis of ds homologous DNA molecule starts to begin
the DNA synapsis

Homologous Genetic Recombination: DNA hybridization reactions model
• When a double helix DNA re-forms from a ssDNA. This is also
called DNA renaturation or hybridization
• This step follows a quickly zipping up the DNA molecule base
pairing to the maximum
• Annealing is required bc the DNA is in unfolded form
• Some times ssDNA strand folds back on itself for the base
pairing like a short hairpin
• This is critical condition for the cells i.e. why Single-Strand
binding protein is required
• This SSB binds and melts out short hairpin helices and let
annealing to start with the complementary strand
• This protein has high affinity to bind with the sugar-PO4
backbone, holding them in correct confirmation while bases
hanging out
• This way the DNA strand can bind to either its complementary
strand (replication) or other DNA strand (recombination)
• In general recombination ssDNA helix should make
complimentary base pairing in dsDNA helix NOT WITH
SINGLE STRAND DNA

Homologous Genetic Recombination: RecA and its Homologs
• Like ssDNA binding protein, RecA binds to dsDNA and ssDNA helix
together
• RecA has multiple DNA binding sites and catalyzes multistep
synapsin formation
• Before this the homology between ssDNA and the region in dsDNA
strand is identified by making transient base pairing
• Once synapsis starts, short heteroduplex regions have begun to
make base pairing to the longer distances via process called branch
migration
• This branch point can occur at any point where two single DNA
strands with the same sequences are attempting to pair with the
same complementary strand
• In this an unpaired region of ssDNA displaces the paired region of
ssDNA, moving branch point having no changes in the DNA nt base
pairing numbers
• Branch migration can occur in both direction, ie.why remains
inefficient
• RecA protein catalyzes unidirectional branch migration, forming fast
heteroduplex DNA with 1000 of base pairs
• RecA is DNA-dependent ATPase, with ATP hyrolyzing site
• RecA tightly binds with DNA+ATP rather than DNA+ADP

Homologous Genetic Recombination: RecA and its Homologs
• ATP continuously added to one end of the RecA protein filaments, while ATP
hydrolyzes to ADP
• Therefore DNA share some dynamics of cytoskeleton filaments actin or tubulin

Homologous Genetic Recombination: Holliday Junction
• Holiday junction contains two of four dsDNA strand
that are crossing and forming the base pairs
• An Holliday junction produces an open,
symmetrical structure
• Further isomerization can interconvert the crossing
and non crossing strands, producing a structure that
is otherwise the same when it starts
• The formation of holliday junction requires ATP
hydrolysis used by sets of proteins
• Two DNA helices with exchanged DNA strand
comes to an end by the process called resolution. In
which exchanged DNA have a cut to release the
exchanged DNA strands
• This release the two identical, homologous DNA
strands, without any alterations in the no. of nt base
pairings
• In the end two original pair of noncrossing strands
is cut resulting two recombinant chromosomes are
formed with reciprocally exchanged main segments
of dsDNA
• Several heteroduplex regions of several thousand
base pairs are readily formed during recombination

Homologous Genetic Recombination: Gene Conversion
• When diploid cell divide into 4 haploid. Each daughter
cells receive half genes from paternal and half genes
from maternal site
• However in rare cases such as in yeast 3 haploid cells
receive 1 of copy of genes from maternal and only 1 cell
receive a copy of genes from paternal allele
• This type of genetic transfer is called as gene
conversion
• This type of conversion is usually seen in meiosis and
less seen in mitosis
• In this conversion only a part of the DNA undergo
conversion, and a part of a gene is changed
Gene Conversion:
• DNA sequence information is transferred from one
DNA helix to another DNA helix whose sequence is now
altered due to the transfer
• It could happen by a homologous recombination
process that juxtaposes two homologous dsDNA helices
OR
• Short piece of DNA synthesis occur for the new allele
base pairing

Homologous Genetic Recombination: Gene Conversion
• Simply a heteroduplex joint forms in which both DNA helices have different nt sequences and are
not matched
• Unmatched sequences are removed by DNA repair mechanism, resulting in the formation of an
extra copy of DNA sequence on the opposite strand
• Then the same gene conversion process occur without crossover
• This requires only the invasion in the dsDNA helix to form heteroduplex region
• These types of gene conversion is responsible for the facile transfer of genetic information
• This type of conversion observed between gene copies in a tandem array of repeated genes

Homologous Genetic Recombination: Outcomes in Meiosis and Mitosis
• Either outcome in general recombination
1- The DNA synthesis involved convert some of the genetic information at the site of the double
stranded break to that of the homologous chromosome
2- If these regions represent different alleles of the same gene
3- Then the nucleotide sequence in the broken helix is converted to that of unbroken helix,
resulting a gene conversion

Homologous Genetic Recombination: Prevention of Promiscuous
Recombination b/w two poorly matched DNA sequences
• Mismatch proofreading system normally
recognizes the mispaired bases in an initial
strand exchange
• If there are significant number of
mismatches then the enzyme maschinery
that breaks and rejoin the two paired DNA
helices are prevented
• These type of mechanism protect bacteria
or cells from invading foreign DNA to form
base pairs with the host DNA
• As vertebrate cells contains many closely
related DNA sequences, same type of
recombination mechanism occurs to prevent
promiscuous recombination
• If not otherwise it would srambled the
genome

Site-Specific Recombination
• It can alter gene order and also add new information to the
genome
• In this recombination the genes position changes within
one chromosomes OR to another chromosomes
• In this specialized nt sequences moves on a
nonhomologous sites within a genome. These movable
sequences are called mobile genetic elements
• These elements differ in size and ranges from 100s or
10,000s nt base pairs
• These elements are present in all cells from E coli. to
humans
• In viruses these moveable elements are required to move
their genomes out from them to the host cell
• Viruses can pack their material in to a viral particles that
can move from one cell to another via ECM environment
• But some viruses cannot move out from the cell in which
they reside due to lacking of intrinsic ability
• 45 % of our genome is composed of these type of mobile
genetic elements
• Due to random mutations only few remains active to move

Site-Specific Recombination
• Importance of this type of recombination is to produce many genetic variants on which
evolution depends
Reason:
These movable variants can also alter the adjacent host cells genome DNA sequences,
by carry them to another site

Site-Specific Recombination: Transpositional or Conservative movement
• Tranpositional Recombination :
1- Site-specific recombination requires enzymes and specific DNA sites
2- Does not involve the formation of heteroduplex DNA
3- It involves the ends of the broken DNA segments in chromosome + these ends should be attached at
one of many different nonhomologous target DNA sites
• Conservative Site-Specific Recombination
1- This involves the formation of a short heteroduplex joint
2- Due to this it requires short same DNA sequences in donor and recipient DNA molecule

DNA Transposones: Capable of injecting mobile genetic
elements in to any DNA sequences
• Transposons, are moveable genetic elements capable of injecting themselves in to many DNA
sites
• Transposase enzyme is encoded by transposon
Mechanism of action: This enzyme first loosen the transposons from the DNA and then insert it
in to the new DNA sites. Homology b/w the site and the end of the DNA is not an issue
• Usually they move very rarely and this is why they are difficult to detect
• They can be divided in to three classes
1- DNA-only transposons
2- Retroviral-like retrotransposons
3- Nonretroviral retrotransposons

DNA Transposons

Functions and specificities of DNA Transposons
1- DNA-only transposons
In this the mobile element DNA is cut out from the donor DNA and joined in the target
DNA by transposase. Hence exists as a DNA all life
2- Retroviral-like retrotransposons
They don’t move directly rather they need RNA polymerase to transcribe the mobile
element sequence into RNA. Then RNA transcriptase synthesize DNA from this RNA
using it as a template. Then this DNA will incorporate in to new DNA sites of the
target DNA, by an enzyme integrase.
3- Nonretroviral retrotransposons
It also requires RNA transcriptase to convert RNA in to DNA. But here RNA is directly
involved in the transposition reaction

DNA-only Transposons
• DNA transposons moves directly by cut and paste
mechanism bc it’s a direct move towards the target
DNA site
• Each subunit of transposons recognize the same
specific DNA sequence at the end of the element
• This creates a DNA loop which brings close both
the ends
• The transposase cuts this DNA loop to expose the
element, hence remove it from the chromosome
• Then transposase attacks on the target DNA mol,
breaking two PO4 diester binds
• While joins the elements in the target DNAs
molecule
• The gap created in the host DNA while moving out
the element, is filled by DNA polymerase and ligated
by DNA ligase
• This produces DNA of variant sizes, which can be
serve as a convenient markers of a prior
transpositional site specific recombination event

DNA-only Transposons
• Some times the gap in the donor chromosome
DNA get filled by nonhomologous end-joining
reaction. This generates a chance of getting a
mutation at the excised DNA site
• Some DNA-only transposons moves by cut an
paste mechanism called replicative transposition.
In this first the DNA replicates itself and a copy of
it transferred to the target DNA sites
• Hence original DNA remains intact and
unchanged

Transpositional Site-specific Recombinant: Viruses
mode of incorporation in to the host DNA
• Transposons are very useful for viruses
such as retroviruses for eg. AIDS
• Its mode of DNA transfer is first synthesize
dsDNA from ssRNA bc of having reverse
transcriptase enzyme
• During infection RNA enters inside the host
DNA and start the transcription from RNA by
the enzyme, which can polymerize DNA by
on either RNA or DNA template
• The sequences on ds DNA ends
recognized by another enzyme known as
integrase
• Integrase activates 3’-OH viral DNA that
directly attack the target DNA but cut and
paste DNA-only transposons

Retroviral-like Retrotransposons Resemble Retroviruses, but Lack a
Protein Coat
• Mode of transfer of DNA to the target cell requires
the transcription of whole transposon (need to be
transfer)
• This produce RNA copy of element (5000 nt)
• This translated in mRNA by the host cell, encodes
a reverse transcriptase enzyme
• Reverse transcriptase synthesize dsDNA from
RNA
• Then integrase enzyme insert the DNA in to the
target chromosome (DNA)
• Integrase enzyme also encoded by the Ty1 DNA
• But Ty1 remains inside the host cells and didn’t
move out bc it lacks a protein coat
• But other Retroviral-like Retrotransposons they
move out and infect many other cells

Nonretroviral Retrotransposons
• Most of the human DNA is composed of LINE element (Long interspersed nuclear element)
• Although most of the copies of L1 element are immobile, a few retain the ability to move
• Some times movement of these elements causes a disease for eg in Hemophilia, L1 insertion in to the
gene encoding blood clotting factor VIII
• Nonretroviral retrotransposons can also be found in Yeast mitochondria, mammals and insects
• They move via the help of endonuclease complex and reverse transcriptase
• Other DNA repeats that lacks either endonucleases or reverse transcriptase in their nt sequences uses
cell’s endonucleases and reverse transciptase, including L1 elements
• For eg Alu elements lacks endonucleases or reverse transcriptase genes, still it has amplified and
becomes the major part of the human genome
• Alu and L1 genes sequences are closely related to the mouse sequences, but their incorporation in mouse
sequences is different than in humans

Reversible rearrangement of DNA = Conservative Site-specific recombination
• It mediates the rearrangements of other
types of mobile DNA elements
• In this breaking and joining occur at two
special sites, one on each of the DNA
strand
• The depending upon orientation of the two
recombination sites
1-DNA integration
2- DNA excision
3- or DNA inversion can occur
In this same enzyme is used to separate
and join the two DNA strand, restoring two
original DNA molecules. These enzymes
have reversible action on the joining and
breaking of the DNA strand

Conservative Site-specific recombination: An example
• Bacteriophage lambda virus infects a bacterial DNA and
synthesize an encoded enzyme integrase
• Viral DNA covalently joins with the bacterial (host)
chromosome, and becomes a part of host DNA and
replicates
• Integrase enzyme act so by recognizing special site on
the host as well as on the viral DNA for the joining of the
two strand
• These sites have different nt sequences but are related
• Recombination starts and requires integrase proteins to
binds tightly with the host and viral DNA. This brings both
DNA closer to each other
• Integrase now cuts and reseal the nick in the DNA strands
at sites specific strand exchange
• Lambda integrase resembles like DNA topoisomerase,
which is required for the formation of the covalent linkages
• Due to this reason this exchange canoccur in the
presence of ATP and Ligase

• To reverse this link between two strands the same
mechanism can be use to excise the DNA
• By getting specific signals from the cells lambda
virus DNA jumps and leaves the sites on
chromosome and multiply rapidly in the bacteria
• This excision is catalyze by excisionase
• Excisionase is only synthesized by the viral DNA
when the bacterial DNA is in stress
• This is a signal for a virus to leave the host cell and
multiply again as a virus particle
Conservative Site-specific recombination: An example

The life cycle of bacteriophage Lambda

Conservative Site-Specific Recombination Can be Used to Turn Genes On or Off

Conservative Site-Specific Recombination Can be Used to Turn Genes On or Off
• When special sites are recognized by the Conservative Site-Specific Recombination
enzymes
• Enzymes are inverted, DNA sequences between them is also inverted rather than
excised
• This type of DNA inversion is used to control gene expression by bacterias
• They can assemble active genes from the separated coding segments
• These segments are inheritable to the daughter cells
• For eg Conservative Site-Specific Recombination enzymes sequences
+
DNA strand containing the sites which is recognized by the Conservative Site-Specific
Recombination enzyme are introduce in a mouse
• After some time the genes for enzyme synthesis are activated to rearrange the target
DNA sites
• This type of rearrangements usually leads to the formation of different proteins in the
tissues of a mouse
• By this we can turn off the genes as well
• This is very useful in knowing the functions of many proteins in particular cells or tissues

Lecture. 5 DNA Recombination

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