Recombination

1. RECOMBINATION
Submitted to: Submitted By:
I.K Nishitha Aleena Stanley
Assistant Prof: 1st MSc Botany
Dept of Botany St.Teresa’s College
St.Teresa’s College
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2. RECOMBINATION
● Recombination is the rearrangement of DNA
molecule or formation of new combination of
genes.
● Recombination by crossing over is the process
most molecular biologists often associate with the
term recombination.
● But crossing over is not only the mechanism for
recombination.
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3. Three Mechanisms by which recombination can take place;
1. Homologous Recombination
2. Non-Homologous Recombination
3. Site specific recombination
4. Transposition
These are important mechanisms for DNA
rearrangement(Recombination)
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4. HOMOLOGOUS RECOMBINATION (Generalized recombination)
It is the process whereby DNA segments that are similar or identical to each other
break and rejoin to form a new combination.
Note: Homologous Recombination- occurs between DNA molecules of very similar or identical
sequence.
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5. Two types of crossing over may occur between replicated chromosomes in a
diploid species:-
1. Sister Chromatid Exchange (SCE)
It Occurs between sister chromatids- genetically identical chromatids- doesn’t
produce new combination of alleles.
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6. 2.Homologous Recombination
It occurs when homologous chromosomes cross over- produce new combination
of alleles; result in genetic recombination
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7. ● It is most widely used by cells to accurately repair harmful
breaks that occur on both strands of DNA, known as
double strand breaks.
● These new combinations of DNA represent genetic
variation in offspring, which in turn enables populations to
adapt during the course of evolution.
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8. Models Explaining Homologous Recombination
1. Holliday Model
2. Meselson- Radding Model
3. Double- strand Break Model
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9. 1.HOLLIDAY MODEL
● Robin Holliday proposed a model in
1964 to explain the molecular steps
that occur during homologous
recombination.
● This model describes a molecular
mechanism of the recombination
process.
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10. Steps in Homologous Recombination
Alignment of
two
homologous
DNA
molecules.
1
Introduction
of breaks in
the DNA.
2
Strand
invasion.
3
Formation of
the Holliday
junction.
4
Resolution of
the Holliday
junction.
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11. Holliday Model-Steps
1. Two homologous chromatids are
aligned with each other.
2. A break or nick occur at identical sites
in one strand of each of the two
homologous chromatids.
3. The strands then invade the opposite
helices and base-pair with the
complementary strands.
4. This event followed by the covalent
linkage to create a Holliday junction.
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12. 5. When the two strands have crossed over and DNA ligase sealed the new
intermolecular phosphodiester bonds, a Holliday Junction is created- also
Known as chiasma or chi structure.
Holliday Junction
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13. 6. A Holliday Junction can move along the DNA by the repeated melting and formation
of base pairs- BRANCH MIGRATION.
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14. 7. Because the DNA sequence in the homologous chromosomes are similar but
may not be identical, the swapping of the DNA strands during branch migration
may produce a heteroduplex.
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15. 8. The final step in the recombination process is called
resolution because they involve the breakage and
rejoining of two DNA strands to create two separate
chromosomes. It is the step to regenerate DNA molecule
and therefore finish genetic exchange.
Two ways a resolution can happen, In this 2DNA’s are recombined
and it can be found in single plain after rotating.
( All the DNA Strands are on the same plane). Here 2 DNA strands
are crossing each other. So to separate the crossing - vertically
and horizontally.
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16. Here the 2 DNA Strands are recombined(each
made of 2 nucleotide seq). So each strands
are crossover strands.
Here in Both of the 2 DNA strand one
strand is conserved. And one strand is
recombined and these products are called
non crossover products.
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17. 2.The Meselson- Radding Model
● Proposed by Mathew Meselson and Charles Radding in
1975.
● Hypothesized that a single nick in one DNA strand initiates
recombination.
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18. ● This model suggests, Single-
strand nick occurs in one of the
double helices, one of the free
end invades the homologous
double helix( unbroken),
displacing one of its strands
forming a D-loop
(Displacement loop)
D loop 18

19. ● Eventually second nick occurs at the
D-loop, creating the Holliday
structure.
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20. ● The Final steps( branch migration
and resolution) is as same as in
Holliday model.
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21. 3. The Double strand Break (DSB) Model
● Proposed by Jack Szostak, Terry Orr-Weaver, Rodney Rothstein and
Franklin Stahl.
● Suggests that a double- strand break initiates the recombination process.
● Recent evidence suggests that double- strand breaks commonly promote
homologous recombination during meiosis and during DNA repair.
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22. ● Formation of a double strand break in one of
the chromosome .
● A small region near the break is degraded,
which generates a single stranded (with 3’0H)
segment that can invade the intact double
helix.
● The strand displaced by the invading segment
forms a structure called displacement loop.
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23. ● After the D-loop is formed, two
regions have a gap in the DNA.
● DNA synthesis occurs in the
relatively short gaps where a
DNA strand is missing.
● This DNA synthesis is called
DNA gap repair synthesis.
● Once this completed, two
Holliday junctions are formed.
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24. Depending on the way these are resolved, the end result is non recombinant
or recombinant chromosomes containing short duplex.
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25. DSB Repair Model
A DNA-cleaving enzyme
sequentially degrades the
broken DNA molecule to
generate regions of
single-stranded DNA
(ssDNA).
Creation of ssDNA tails
which terminate with
3' ends.
The invading strand
base-pairs with its
complementary strand
in the other DNA
molecule.
Introduction of a DSB
in one of two
homologous duplex
DNA molecules.
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26. The invading strands
with 30 termini serve as
primers for new DNA
synthesis.
Elongation from these
DNA ends using the
complementary strand in
the homologous duplex
as a template.
Gene conversion event.
The two Holliday junctions
found in the recombination
intermediates generated by
this model move by branch
migration.
Resolution.
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27. Homologous Recombination Protein Machines
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28. RecA Protein
● RecA Protein is about 350 amino acids residues. Its sequence is highly conserved
among eubacterial species.
RecA protein involved in homologous recombination and bypass mutagenic DNA
lesions by SOS response.
1) ATP-driven homologous pairing and strand exchange of DNA molecules necessary for
DNA recombination repair.
2) ATP-dependent uptake of single stranded DNA by duplex DNA
3) ATP-dependent hybridization of homologous single-stranded DNAs.
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29. The RecBCD Helicase/Nuclease
• Processes broken DNA molecules to generate these regions of ssDNA.
• Helps load the RecA strand-exchange protein onto these ssDNA ends.
• Multiple enzymatic activities of RecBCD provide a means for cells to
“determine” whether to recombine with or destroy DNA molecules that
enter a cell.
• Has both DNA helicase and nuclease activities.
• The complex binds to DNA molecules at the site of a DSB and tracks along
DNA using the energy of ATP hydrolysis.
• The DNA is unwound, with or without the accompanying nucleolytic
destruction of one or both of the DNA strands.
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30. • composed of three subunits (the products of the recB, recC, and recD genes)
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31. ● The Rec B protein contains 1180 residues and is modular .
● The N terminal contains the helicase activity and has seven characteristic SF1 motifs 1,1a, 2, 3,
4,5 and 6
● The C terminal domain contains nucleases motifs. “Nuc” marks the position of nuclease activity
contains aspartate and lysine residues.
● The Rec C protein has 1122 aa residues and contains chi recognition site.
● The Rec D protein with 608 aa residues has SF1 seven helicase motifs. (1, 1a, 2, 3, 4, 5
and6 )
Rec B
Rec C
Rec D
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32. RecBCD-catalyzed DNA end-processing reaction.
● DNA ends resulting from a double- strand break is
processed by a multi functional enzyme complex
called RecBCD.
● RecBCD is a sequence- regulated bipolar helicase
nuclease that splits the duplex into its component
strands and digests them until it encounters Chi site.
● Chi site - Recombination Hotspot
● (5’-GCTGGTGG-3’)
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33. 1) The RecBCD binds tightly to a blunt DNA end of
a linear DNA duplex.
2) RecBCd couples the hydrolysis of ATP to DNA
translocation and unwinding. The ssDNA
products are cleaved asymmetrically, with the
degradation on the 3’- terminated ssDNA tail
being much more vigorous than the degradation
of the complementary tail.
3) The enzyme continues the translocation until it
pauses at a correctly oriented Chi sequence.
After Chi sequence recognition RecBCD
facilitates the loading of the RecA protein onto
the 3’ ssDNA tail.
4) The enzyme continues to translocate, but the
nuclease polarity is switched; the degradation of
3’ ssDNA tail is attenuated, whereas the
hydrolysis of the 5’ ssDNA tail is upregulated. 33

34. 5) RecBCD repeatedly deposits RecA
promoters, which act as nucleation points for
filament growth primarily in the 5’-3’ direction.
6) RecBCD enzyme dissociates from the DNA .
The product of the enzyme is recombinogenic
nucleoprotein complex of the RecA protein
bound to the 3’ ssDNA tail with Chi at its
terminus.
The product invade homologous duplex DNA to
promote the recombinational repair of a DSB or
to restart DNA replication.
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35. Non Homologous Recombination
● Non-homologous recombination
refers to a DNA rearrangement
that leads to covalent joining of
non-homologous linear DNA
segments.
● Occurs in most gram positive
bacteria.
● E.g Kluyveromyces lactis
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36. ROLE OF Ku PROTEIN
● Ku is abundant, highly conserved DNA binding protein found in both
prokaryotes and eukaryotes that play essential roles in the maintenance of
genome integrity.
● In eukaryotes, Ku is a heterodimer comprised of two subunits, Ku70 and Ku80
that is best characterized for its central role as the initial DNA end binding
factor in the “classical” C-NHEJ pathway.
● Ku binds dsDNA ends with high affinity in a sequence-independent manner
through a central ring formed by the intertwined strands of the Ku70 and Ku80
subunits.
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37. Canonical non-homologous end joining pathway(C-NHEJ)
C-NHEJ depends on Ku heterodimer and DNA- PK
catalytic subunit( DNA-PKcs), which together form the
DNA -PK holoenzyme.
Unlike MRN, which can bind internally, Ku requires a
free DNA end for binding and cannot associate with
most blocked ends.
Several nucleases including (TDP1/2) and Artemis can
remove hairpins, damaged bases or proteins -DNA
adducts.
The DNA ends are processed by additional enzymes
and rejoined by the LIG4/XRCC4/XLF complex.
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38. Corresponding Enzymes in Prokaryotic and Eukaryotic NHEJ Components.
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