• Maintaining a low mutation rate is essential for cell viability and health. It is estimated that both in prokaryotic and eukaryotic cells, DNA is replicated with very high fidelity with one wrong nucleotide incorporated once per 108–1010 nucleotides polymerized. The fidelity of DNA replication relies on nucleotide selectivity of replicative DNA polymerase, exonucleolytic proofreading, and post-replicative DNA repair systems. • Mutations can occur due to errors in DNA replication as well as due to certain damages to the DNA. Errors in replication are corrected to a great extent by proofreading mechanisms. Maintaining the genetic stability that an organism needs for its survival requires not only an extremely accurate mechanism for replicating DNA but also mechanisms for repairing many accidental lesions that occur continually. Most such spontaneous changes in DNA are temporary because they are immediately corrected by a set of processes that are collectively called DNA repair.• Maintaining a low mutation rate is essential for cell viability and health. It is estimated that both in prokaryotic and eukaryotic cells, DNA is replicated with very high fidelity with one wrong nucleotide incorporated once per 108–1010 nucleotides polymerized. The fidelity of DNA replication relies on nucleotide selectivity of replicative DNA polymerase, exonucleolytic proofreading, and post-replicative DNA repair systems. • Mutations can occur due to errors in DNA replication as well as due to certain damages to the DNA. Errors in replication are corrected to a great extent by proofreading mechanisms. Maintaining the genetic stability that an organism needs for its survival requires not only an extremely accurate mechanism for replicating DNA but also mechanisms for repairing many accidental lesions that occur continually. Most such spontaneous changes in DNA are temporary because they are immediately corrected by a set of processes that are collectively called DNA repair.• Maintaining a low mutation rate is essential for cell viability and health. It is estimated that both in prokaryotic and eukaryotic cells, DNA is replicated with very high fidelity with one wrong nucleotide incorporated once per 108–1010 nucleotides polymerized. The fidelity of DNA replication relies on nucleotide selectivity of replicative DNA polymerase, exonucleolytic proofreading, and post-replicative DNA repair systems. • Mutations can occur due to errors in DNA replication as well as due to certain damages to the DNA. Errors in replication are corrected to a great extent by proofreading mechanisms. Maintaining the genetic stability that an organism needs for its survival requires not only an extremely accurate mechanism for replicating DNA but also mechanisms for repairing many accidental lesions that occur continually. Most such spontaneous changes in DNA are temporary because they are immediately corrected by a set of processes that are collectively called DNA repair.

1. Seminar Presentation-Semester 2
Subject: Molecular Biology
Topic: Recombination in Repair and Damage of DNA
Submitted to,
Dr. Giby Kuriakose
Head of the Dept. Of Botany
Submitted by,
Anakha Mariya Jacob
21PBOT2476

2. Recombination
in Repair and Damage
of DNA

3. Fidelity of DNA
Replication
• Essential for cell viability and health.
• High fidelity: 1 wrong nucleotide/
10^8-10^10 nucleotides
polymerized.
• Nucleotide selectivity of replicative
DNA polymerase, exonucleolytic
proofreading, and post replicative
DNA mismatch repair (MMR).
https://th.bing.com/th/id/R.1cd5238578f5a7b4f70fad891fef8a59?rik=M3kFKlYeSbooUQ&riu=http%3a%2f%2fwallpapercave.com%2fwp%2fwc169
5740.jpg&ehk=63GxdM%2b55XToGPGuYNLWmM%2fxNVg5gfOfRR2MvxA5XLY%3d&risl=&pid=ImgRaw&r=0

4. Errors in
Replication
DNA
Damage
Mutations
DNA Repair
System

5. Causes of Damage of DNA
https://th.bing.com/th/id/R.5b33365546acc798335221da45cc0b66?rik=zTe2BUhw%2bsuMOA&riu=http%3a%2f%2fkomorlab.ucsd.edu%2fimages%2fDNA_Dam_Out.jpg&ehk=ydgDoT09NTtSkGwLEC%2bfcCzLtwYHDsiZY42TqU5%2bQoE%3d&risl=&pid=ImgRaw&r=0

6. Types of DNA Damages
Endogenous DNA
Damages
• Oxidation of bases
• Alkylation of bases
• Hydrolysis of bases
• Bulky adduct formation
• Mismatch of bases
Exogenous DNA Damages
• UV-B light
• UV-A light
• Ionizing radiations
• Thermal disruption
• Industrial chemicals

7. Double-Strand
Breaks in the
DNA
• Much less frequent.
• One of the most cytotoxic forms of lesions.
• Phosphate backbones of the two complementary
DNA strands are broken simultaneously.
• Discontinuity in the genetic code.
• Broken DNA ends are also vulnerable to further
physical and chemical assault resulting in lost or
damaged bases or the formation of abnormal
DNA structures, all of which can result in loss of
genetic information.
(https://scfh.ru/files/medialibrary/ebf/ebf8a1b54b76b378edbe1f96b3e4fe59.jpg)

8. Causes of DSB
Clastogens
Ionizing radiation
(IR)
Anticancer
chemotherapeutic
drugs

9. •Produce radiolysis radicals.​
•Attack the sugar-phosphate backbone.​
•10 SSBs for each DSB.​
•Leaves “dirty ends,” consisting of phosphoglycolates and
terminal nucleotides.
Ionizing Radiation
(https://www.researchgate.net/profile/Adam_Smolinski/publication/342459901/figure/download/fig3/AS:909155309219841@1593770925104/The-main-sources-of-ionizing-radiation-and-their-contribution-to-humans-as-
worldwide.png)

10. Anticancer Chemotherapeutic Drugs
https://th.bing.com/th/id/R.bb916edb38740c11a9d5670a006ca359?rik=zXFp7BgrMXpTxg&riu=http%3a%2f%2fdrugline.org%2fimg%2fdrug%2f9144_9220_2.jpg&ehk=vNb61EsLUS%2bjXoXNVqW9k07QVxR55unJnLVF4kgHahw%3d&risl=&pid=ImgRaw&r=0, https://5.imimg.com/data5/HI/H
L/MY-28890488/2-500x500.jpg, https://sep.yimg.com/ay/yhst-12533177228474/mitomycin-injection-sdv-5mg-15ml-vl-accord-16729011505-
30.gif, https://api.meithealpharma.com/SavedFiles/UnitSmallImage_9_99a66e1.jpg , https://th.bing.com/th/id/OIP.dBVvdC81zBX19uN2QCUolAHaHa?pid=ImgDet&rs=1

11. Repair of DSB
DNA end-joining
Non homologous end-
joining (NHEJ)
Alternative end
joining/microhomology
mediated end joining
Homologous
recombination
V(D)J recombination
Class switch
recombination

13. Thymine dimers
• Effect of thymine dimers on DNA replication.
• Replication fork is temporarily stalled.
• Hydrogen bonding with two adenine bases.
• Distortion in the DNA helix.
• Polymerase adds adenine to the growing chain.
• reacts to the distortion, thereby removes the added adenine
by its proofreading activity.
• Cycle of adenine addition and removal
• Polymerase stalls at this site.
• ‘post-dimer initiation’ and ‘trans-dimer synthesis’

14. Homologous Recombination
• Occurs in newly replicated DNA.
• Used only during and shortly after DNA replication (in S and G2 phases).
• Sister chromatid as a template.
• DSBs also arise from DNA replication forks that become stalled or broken.

16. 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.
5

17. Step 1: Alignment of two homologous DNA
molecules
• The replicated daughter strands are different in nature.

18. Step 2: Introduction of breaks in the DNA
• Processing of the ends at the breaks to generate regions of single-
stranded DNA.

19. Step 3: Strand invasion
• Initial short regions of base pairing are formed between the two
recombining DNA molecules.
• Generation of heteroduplex DNA.
• Strand-exchange proteins & sister-strand exchange.
• generates a Holliday junction

20. Step 4: Formation of the Holliday junction
• The two DNA molecules become connected by crossing DNA strands
to form Holliday junction.
• Can move along the DNA by the repeated melting and formation of
base pairs.
• Branch migration.

21. Step 5: Resolution of the Holliday junction
• Resolution
• Either by cleavage of the Holliday junction or (in eukaryotes) by a
process of “dissolution.”
• Two distinct classes of DNA products.

22. Splice recombination
products
Patch products
• crossover product. • non-crossover
products.
• composed entirely of
DNA from one of the
two parental DNA
molecules.
• contain regions of
sequence from both
parental molecules.
• results in
reassortment of
genes that flank the
site of
recombination.
• does not result in
reassortment of the
genes flanking the
site of initial
cleavage

23. Double-strand break –repair pathway
Introduction of a DSB in
one of two homologous
duplex DNA molecules.
A DNA-cleaving enzyme
sequentially degrades the
broken DNA molecule to
generate regions of single-
stranded DNA (ssDNA).
Creation of ssDNA tails
which termianate with 3'
ends.
The invading strand base-
pairs with its
complementary strand in
the other DNA molecule.

24. 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.

25. Homologous Recombination Protein
Machines

26. 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.
• composed of three subunits (the products of the recB, recC, and recD genes)
• 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.
• Chi sites (for “crossover hot spot instigator”)- stimulate the frequency of
homologous recombination.
• The RecB and RecD subunits are both DNA helicases.

27. RecBCD
Helicases/
Nucleases
• RecB subunit contains a 30 -to-50 helicase &
a multifunctional nuclease domain that
digests the DNA as it moves along.
• RecD is a 50 -to-30 helicase.
• RecC functions to recognize Chi sites.
• Chi sites within DNA act as a sort of
“molecular throttle” to regulate the
activities of the helicases and therefore the
speed of DNA translocation.

28. The looped-out ssDNA is
pulled or reeled back in by
the RecB subunit, and RecB
becomes the primary motor
now leading the complex.
Conformational change
occurs that results in
uncoupling of the RecD
subunit.
The nuclease activity of the
RecBCD complex is altered.

29. Chi Sites Control RecBCD
• Increase the frequency of recombination 10-fold.
RecA Protein
• strand-exchange proteins.
• The active form of RecA is a protein–DNA filament.
• The filament can accommodate one, two, three, or even four strands of DNA.
• To form a filament, subunits of RecA bind cooperatively to DNA.
• The filament grows by the addition of RecA subunits in the 50 -to-30 direction.

30. Assembly of RecA occurs on a molecule
containing a region of ssDNA, such as an ssDNA
tail.
RecA–ssDNA complex participates in the search
for a homology.
Once a region of base-pair complementarity is
located, RecA promotes the formation of a stable
complex with complete Watson–Crick hydrogen
bonding between these two DNA molecules.
RecA-bound three-stranded structure called a
joint molecule is formed.
The DNA strand in the primary binding site
becomes base-paired with its complement in the
DNA duplex bound in the secondary site.

31. RuvAB Complex
• Specifically Recognizes Holliday Junctions and Promotes Branch
Migration.
RuvA recognizes and
binds to Holliday
junctions and recruits
the RuvB protein to this
site.
RuvB ATPase provides
the energy to drive the
exchange of base pairs
that moves the DNA
branch.

32. RuvC
• RuvC recognizes the Holliday junction in a complex
with RuvA and RuvB and specifically nicks two of
the homologous DNA strands that have the same
polarity.
• This cleavage results in DNA ends that terminate
with 50 -phosphates and 30 -OH groups that can
be directly joined by DNA ligase.
• Cleavage takes place only at sites conforming to
the consensus 50 -A/T-T-T-G/C.
• Cleavage occurs after the second T in this
sequence.

33. The RecBCD binds to linear DNA at
a free (broken) end and moves
inward along the double helix,
unwinding and degrading the DNA
in a reaction coupled to ATP
hydrolysis.
The enzyme nears a sequence
called chi (5′) GCTGGTGG.
From this point, the degradation of
the strand with a 3′ terminus is
greatly reduced but degradation of
the 5′ terminal strand is increased.
This process creates single-stranded
DNA with a 3′ end.
The RecA monomers assemble
cooperatively on DNA and form a
helical filament.
The RecF, RecO and RecR proteins
regulate the assembly of RecA
filaments.
As the duplex DNA is incorporated
within the RecA filament and
aligned with the bound single-
stranded DNA, one strand of the
duplex switches pairing partners.
Continued strand exchange requires
an ordered rotation of the two
aligned DNAs. This results in a
spooling action that shifts the
branch point along the helix. Results
in ‘Holliday intermediate’
Once a Holliday intermediate is
formed, enzymes such as
topoisomerases, the RuvAB branch
migration protein, resolvase,
nuclease, polymerase and ligase
complete the recombination
reaction.
The RuvC protein of E. coli cleaves
Holliday intermediates to generate
full-length and unbranched
recombined and repaired
chromosome products.

34. Recombination repair in eukaryotes
• The RAD 52 group of genes is required for recombination repair in
eukaryotes.
• The MRX (yeast) or MRN (mammals) complex is required to form
single-stranded region at each DNA end.
• The RecA homologue Rad51 forms a nucleoprotein filament on the
single-stranded regions, assisted by Rad 52 and Rad 55/57.
• Rad 54 and Rdh54/Rdh54B are involved in homologue search and
strand invasion.
• Following repair synthesis, the resulting structure is resolved

35. Homologous
Recombination in
relation to Cancer
• HR deficiency leads to genetic
instability, and, as expected,
many HR genes are
downregulated in cancer cells.
• The link between HR deficiency
and cancer predisposition is
exemplified by familial breast
and ovarian cancers and by
some subgroups of Fanconi
anaemia syndromes.
• ‘RAD51 paradox’.

36. • Mutations in HR genes
BRCA1, BRCA2, PALB2,
BARD1, RAD51C, and
RAD51D correlate with
increased breast cancer
incidence.
• AKT1, which is
negatively regulated by
PTEN (one of the genes
mutated in hereditary
breast and ovarian
cancer), also inhibits HR
through the
cytoplasmic
sequestration of BRCA1
and RAD51, resulting in
at least a BRCA1
defective-like
phenotype.

38. Reference
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10.1016/j.dnarep.2010.09.018.
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DNA repair.” Cold Spring Harbor perspectives in biology vol. 6,9 a016428. 7 Aug. 2014,
doi:10.1101/cshperspect.a016428.
3. Chmielewska, Izabela & Chałupnik, Stanislaw & Wysocka, Małgorzata & Smoliński, Adam. (2020). Radium
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34316706; PMCID: PMC8209977.