NAME: WAFA UM-UL BANEEN

ROLL NO. 18BSZOL28765

SUBJECT : Zoology
Seminar Topic :
DNA DAMAGE AND IT’S REPAIRING MECHANISM
 OUTLINE:
 What is DNA?
 Functions of DNA
 DNA Damage
 Types of DNA Damage
a. Oxidative processes
b. Alkylation of bases
c. Base loss caused by hydrolysis of bases.l
d. DNA crosslinked
e. UV Radiation
 DNA repairing mechanism
 Types of DNA repairing :
1.Direct reversal
a.photoreactivation repair
b.methylgroup removal
2.Single Strand Damage
a.BER
b. NER
c.MMR
3.Double strand break
a.NHEJ
b.MMEJ
c.homologous recombination
 Diseases associated with DNA repairing system:
a. Bloom syndrome
b. Cockayne’s syndrome
c. Werner syndrome
d. Progeria
e. Rothmund Thomson syndrome
 DNA DAMAGE AND IT’S REPAIRING MECHANISM
 WHAT IS DNA?
 DNA stand for “DEOXYRIBONUCLEIC ACID”
 Deoxyribonucleic acid is a polymer made of two polynucleotide chains that coil around
one another to form a double helix and which contains the genetic material necessary for
all known creatures, including many viruses, to todevelop, function, grow, and reproduce.
 Nucleic acids include DNA and ribonucleic acid.
 By dividing into two single strands, each of which acts as a template for a new strand,
DNA replicates.
 The identical hydrogen-bond pairing between bases that occurs in the double helix is
used to copy the new strands. One of the original strands and one new strand are each
present in each of the two newly formed double-stranded DNA molecules.
 FUNCTION OF DNA:
 A transition from B-DNA to A-DNA occurs during transcription.
 A-DNA also play important role in some processes that donot involve RNA.
 DNA is a molecule that is present in almost all cells and contains the instructions
needed to make proteins, which are unique molecules vital to the growth and operation
of the body.
 DNA DAMAGE :
 DNA damage is a modification to the DNA’s fundamental structure that does not occur
during DNA replication. A DNA damage can be either a break in one or both of the
links that connect the DNA strands or a chemical addition or disruption to a DNA base
that results in a nucleotide fragment.
 The primary structure of the double helix is most frequently impacted by DNA damage,
which results in chemical modifications to the bases. By adding foreign chemical bonds
or large adducts that do not fit in the typical double helix, these modifications have the
potential to alter the molecules’ normal helical structure. DNA typically doesn’t have
tertiary structure, unlike proteins and RNA, so disruption or damage cannot happen
there. However, in eukaryotes, DNA is wrapped around “packaging” proteins called
 histones and supercoiled, both of which superstructures are susceptible to the effects of
DNA damage.
 TYPES OF DNA DAMAGE:
There are several types of DNA damage that can occur due either to normal cellular
processes or due to the environmental exposure of cells to DNA damaging agents. DNA
bases can be damaged by:
(1) oxidative processes
(2) alkylation of bases
(3) base loss caused by the hydrolysis of bases
(4) DNA crosslinkand

1. OXIDATIVE PROCESSES:
 Reactive oxygen species (ROS) can cause significant cellular stress and damage
including the oxidative damage of DNA. Hydroxyl radicals (•OH) are one of the most
reactive and electrophilic of the ROS and can be produced by ultraviolet and ionizing
radiations or from other radicals arising from enzymatic reactions.

 According to estimates, each cell’s DNA may experience up to 100,000 8-oxo-dG

lesions per day. Guanosine has a lower reduction potential than 8-oxo-dG.As a result, it
can undergo further oxidation, producing a range of secondary oxidation products.
  As a biomarker of oxidative stress, elevated levels of 8-oxo-dG in a tissue can be used.
Additionally, elevated levels of 8-oxo-dG are frequently discovered in connection with
the development of cancer and other illness conditions.Thus, 8-oxo-dG mutations
typically result in a G to T transversion.
2. ALKYLATION OF BASES:
 Alkylating substances are abundant in the environment and are also created internally
by cells as waste products of metabolism. They cause cytotoxic, mutagenic, or neutral
lesions to be introduced into DNA or RNA bases.
 Methylation is the most prevalent type of alkylation, and the main byproducts are N7-
methylguanine (7meG), N3-methyladenine (3meA), and O6-methylguanine (O6meG).
O4-methylthymine (O4meT), N1-methyladenine (1meA), N3-methylcytosine (3meC),
and methyl phosphotriesters are among the other DNA bases that experience minor
methylation (MPT).
3. BASE LoSs:
 An AP site (apurinic/apyrimidinic site), also known as an abasic site, is a region of
DNA (and, much less frequently, RNA) that neither naturally contains a purine base
nor a pyrimidine base due to DNA damage.
 AP sites can develop spontaneously by depurination, but they can also develop as a
result of the base excision repair process.
 AP sites can cause mutations during semiconservative replication if they are not
corrected. They are frequently avoided by translesion synthesis and can result in
replication fork stalling.
4. DNA CROSSLINKED:
DNA crosslinking happens when different exogenous or endogenous substances interact
with two DNA nucleotides, creating a covalent bond between them. Within the same
strand of double-stranded DNA (intrastrand) or between the opposing strands
(interstrand), this crosslink can take place .
5. UV Radiation:
 Indirect DNA damage is caused by UVA and UVB radiation when photons are absorbed
by chromophores that are not DNA. As a result, reactive oxygen species are produced,
including singlet oxygen and hydrogen peroxide, which damage DNA bases and result in
mutations.
 DNA REPAIRING:
 The integrity of a cell’s genetic code is maintained by one of numerous methods,
including DNA repair. The ability of kids to inherit parental DNA as faithfully as
possible thanks to DNA repair assures the survival of a species. Furthermore, it protects
a person’s health. Cancer and other genetic illnesses can be brought on by changes in the
genetic code.
 The majority of DNA damage is repaired by removing the harmed bases and then
resynthesising the excised area. However, some DNA lesions can be directly reversed,
which may be a more effective method of handling some types of DNA damage that
happen frequently.
 The type of cell, the cell’s age, and the extracellular environment are only a few of the
variables that affect how quickly DNA is repaired. One of three situations is feasible for
a cell that has sustained significant DNA damage or that is no longer capable of properly
repairing DNA damage Or one that is no longer capable to properly repairing DNA
damage, can go into one of two states: An unreversible state of dormancy that can result
in the growth of a malignant tumour, cell suicide, also known as apoptosis or
programmed cell death, and uncontrolled cell division
 an organism’s proper operation depends on a cell’s capacity for DNA repair in order for
its genome to be intact. Numerous genes that were once believed to affect lifespan have
now been found to play a role in DNA damage repair and protection.
 DNA REPAIRING MECHANISM:

If DNA damage compromises the integrity and accessibility of crucial information in the
genome, cells cannot operate (but cells remain superficially functional when non-essential genes
are missing or damaged).

There are many different repair techniques that have developed to replace lost information,
depending on the kind of damage done to the double helix shape of DNA. Cells attempt to
restore the original information by using the sister chromatid or the unaltered complementary
strand of DNA as a template. In the absence of a template, cells fall back on translesion
synthesis, an error-prone recovery technique

 TYPES OF DNA REPAIRING MECHANISM

There are different types of DNA Repairing mechanism
1) Direct reversal

A. photoreactivation repair

B. methyl group removalist

2) Single strand break repair

a. base excision repair (BER)
b. Nucleotide excision repair (NER)
c. Mismatch repair (MMR)
3) Double strand break repair
a. non homologous end joining (NHEJ)
b. microhomology mediated end joining (MMEJ)
c. homologous recombination
d.
1) DIRECT REVERSAL :

The damaged area or lesion is directly repaired by specialised proteins in our body in a
process known as direct reversal repair. It is the most straightforward and energy-efficient
approach of DNA repair. Unlike the other single-strand repair process, it doesn’t call for a
 reference template. The damaged area or lesion is directly repaired by specialised proteins in
our body in a process known as direct reversal repair.

 It is the most straightforward and energy-efficient approach of DNA repair. Unlike the other
single-strand repair process, it doesn’t call for a reference template.

 A form of repair called direct reversal repair involves the body’s unique proteins directly
repairing the damaged area or lesion. It is both the most straightforward and energy-efficient
way to repair DNA. The other single-strand repair technique needs a reference template,
whereas this one does not. Additionally, the DNA’s phosphodiester backbone is not broken
during this procedure.
a. Photoreactivation repair
 Photoreactivation is possibly the simplest DNA repair mechanism currently known.
Pyrimidine dimers introduced into DNA by UV irradiation can be repaired by the action of a
single photoreactivating enzyme using light in the range of 310–500 nm as an energy
source.
 Photoreactivation is a DNA repair pathway present in all organisms apart from placental
mammals. During photoreactivation, the enzyme photolyase uses visible light (violet/blue)
 as a source of energy (ATP independent) to repair UV-induced DNA damage in the form of
CDS.
 Photoreactivating enzyme repairs UV damage to DNA by utilizing the energy of
near-UV/visible light to split pyrimidine dimers into monomers.

b. Methyl group removal:

 Demethylation is the chemical process resulting in the removal of a methyl group (CH3)
from a molecule. A common way of demethylation is the replacement of a methyl group by
a hydrogen atom, resulting in a net loss of one carbon and two hydrogen atoms. The
counterpart of demethylation is methylation.
 The 6 methyl group from methylguanine is removed by the DNA repair enzyme known as 6
methylguanine methyltransferase (MGMT). By methylating DNA base pairs, alkylating
chemotherapy drugs like temozolomide act to obstruct DNA replication.

2. SINGLE STRAND BREAK REPAIR:

One of the most significant repair mechanisms in cellular response to camptothecin and
its therapeutically relevant derivatives is the DNA single strand break repair (SSBR)
pathway . As a result, it has been determined that the ability to control SSBR is a viable
way to foretell the development of drug resistance.
 DNA single-strand breaks (SSBs) are breaks in just one of the two strands of the double
helix and are frequently accompanied by damaged or mismatched 5′- end/or 3′-termini.
a. BER:
 Stands for Base excision repair.
 Base excision repair is a cellular mechanism, studied in the fields of biochemistry and
genetics, that repairs damaged DNA throughout the cell cycle. It is responsible primarily for
removing small, non-helix-distorting base lesions from the genome.
 Small base lesions that do not appreciably alter the DNA helix structure can be repaired
using base excision repair (BER). Usually, deamination, oxidation, or methylation causes
such damage.
 Small base lesions that do not appreciably alter the DNA helix structure can be repaired
using base excision repair (BER). The process is started by a DNA glycosylase that detects
the broken base and removes it, leaving an abasic site that can then be processed by either
short-patch or long-patch repair. Different proteins are mostly used by short-patch repair
and long-patch repair to finish BER. BER can occur in mitochondria or nuclei and typically
makes use of distinct protein isoforms or genetically distant proteins. For instance,
apurinic/apyrimidinic site endonuclease 1 (APE1) is significantly more abundant in nuclei
than in mitochondria, whereas APE2 is more abundant in mitochondria.
b. NER:
 Stand for nucleotide excision repair.
 Nucleotide excision repair is a mechanism in which a damaged region of DNA is cut out
and replaced by DNA synthesized using the undamaged strand as template.
 Recognition of the damage leads to removal of a short single-stranded DNA segment that
contains the lesion. The undamaged single-stranded DNA remains and DNA polymerase
uses it as a template to synthesize a short complementary sequence. Final ligation to
complete NER and form a double stranded DNA is carried out by DNA ligase. NER can
be divided into two subpathways: global genomic NER (GG-NER or GGR) and
transcription coupled NER (TC-NER or TCR). The two subpathways differ in how they
recognize DNA damage but they share the same process for lesion incision, repair, and
ligation.
 Remove the bulky DNA lesions that distort the double helix.An enzyme complex
recognizes the distortion resulting from damage.Additional enzymes separate the two
nucleotide strand at damaged region and single strand binding proteins stabilize the
separated strands .

 The sugar phosphate cleaved on both sides of damage.Part of damage is peeled away and
gap is filled by DNA polymerase and sealed by DNA ligase.

c. MMR:

 Stands for Mismatch repair.

 DNA mismatch repair is a system for recognizing and repairing erroneous insertion,
deletion, and mis-incorporation of bases that can arise during DNA replication and
recombination, as well as repairing some forms of DNA damage.
 DNA mismatch repair (MMR) recognizes and repairs erroneous insertion, deletion, and
mis-incorporation of bases that can arise during DNA replication and recombination, and
 repair some forms of DNA damage. It plays an important role in maintaining genomic
stability and cellular homeostasis
 MMR proteins are nuclear enzymes, which participate in repair of base-base mismatch
that occur during DNA replication in proliferating cells. The proteins form complexes
(heterodimers) that bind to areas of abnormal DNA and initiates its removal.
 A mismatch is detected in newly synthesized DNA.
 The new DNA strand is cut, and a patch of DNA containing the mispaired nucleotide and
its neighbors is removed.
 The missing patch is replaced with correct nucleotides by a DNA polymerase.
 A DNA ligase seals the remaining gap in the DNA backbone.
 Many incorrectly inserted nucleotides detected by proofreading are corrected by MMR.
 Enzyme cut out the distorted section of newly synthesized strand of DNA and replace it
with new nucleotides.
 The protein that carried out this in E.coli differentiate between old and new strands of
DNA by the presence of methyl group.
 Adenine nucleotide in GATC sequence is methylated.
 The mismatched repair complex brings the mismatch bases close to the methylated GATC
sequence and new strand is identified.
 Exonuclease removes nucleotide on the new strand between GATC sequence and the
mismatched.
 DNA polymerase then replace the nucleotides correcting the mismatch and Dna ligase
seals the nick in sugar phosphate backbone.
3. DOUBLE STRAND BREAK:
 Double-strand breaks (DSBs) in DNA form as a result of exposure to exogenous
agents such as radiation and certain chemicals, as well as through endogenous
processes, including DNA replication and repair.
 Double-strand DNA breaks are common events in eukaryotic cells, and there are
three major pathways for repairing them:
 homologous recombination (HR) and
 nonhomologous DNA end joining (NHEJ)
 Microhomology-mediated end joining (MMEJ)
ii) Homologous Recombination:
 Homologous recombination repair is a DNA repair process that includes the
invasion of an undamaged DNA molecule by a damaged molecule of identical or
very similar sequence. Resynthesis of the damaged region is accomplished using
the undamaged molecule as a template.
 Recombinase as a RecA bind to the ss-DNA
 Here’s the broken ends are repaired using the information on the intact sister
chromatids or on homologous chromosomes.
 Two of the proteins used in homologous recombination are encoded by Genes
BRCA1 and BRCA2.
  Accessory Factor as Rad54, Rad54B and Rdh54 help recognize and invade the
homologous region
 After the formation of D-loop, the polymerase involved to elongate the

3’invading single strand.

iii) non homologous DNA end joining(NHEJ):
 Non-homologous end joining is a pathway that repairs double-strand breaks in DNA.
NHEJ is referred to as “non-homologous”because the break ends are directly ligated
without the need for a homologous template, in contrast to homology directed repair,
which requires a homologous sequence to guide repair.
 Non homologous end joining is used at other joint of the cell.
  Cycle when sister chromatids are not available for use as HR template when these
breaks occur the cell has not yet replicated DNA that contain the breaks, so unlike
the HR pathway there is no no corresponding template strand available.
 Direct joining of the broken ends. This requires protein thst recognize and bind to
exposed ends and bring them together for ligating . Ku is essential for NHEJ.
c. Microhomology-mediated end joining (MMEJ):
 One of the mechanisms for repairing DNA double-strand breaks is microhomology-
mediated end joining, referred to as alternative nonhomologous end joining.
 Microhomology-mediated end joining (MMEJ) is an error-prone repair mechanism
that involves alignment of microhomologous sequences internal to the broken ends
before joining, and is associated with deletions and insertions that mark the original
break site, as well as chromosome translocations.
 In MMEJ, repair of the DSB is initiated by end resection by the MRE nuclease,
leaving single stranded overhangs. These single stranded overhangs anneal at
microhomologies, which are short regions of complementarity, often 5–25 base
pairs, between the two strands.
 Following annealing, any overhanging bases (flaps) are removed by nucleases such
as Fen1 and gaps are filled in by DNA polymerase theta.This gap-filling ability of
 polymerase theta helps to stabilize the annealing of ends with minimal
complementarity.
 Besides microhomology footprints, polymerase theta’s mutational signature also
consists of (infrequent) templated inserts, which are thought to be the result of
aborted template-dependent extension, followed by re-annealing at secondary
homologous sequences.

 Diseases associated with defective DNA Repairing system:

 Following are the diseased associated with DNA Repairing system …

a. Bloom Syndrome

b.Cockyane’s syndrome

c.Werner syndrome

d.Progeria

e. Rhothmund Thomson syndrome

a. Bloom Syndrome:
 head is dispropotionately small.
 Striking paucity of subcutaneous fat tissues throughout infancy and childhood and
 A redness of th cheeks and nose that charactistically makes it appearance in infancy
after sun exposure.
  Chronic obstructive lung disease, Diabetes mellitus and malignancies of varied type
are some of common complications of Bloom syndrome .
b. Cockayne’s syndrome:
 Cockyane syndrome is a rare autosomal recessive congenital disorder characterized
by growth failure , impaired development of nervous system
and premature aging.
 Hearing loss and eye abnormalities are other common features but problem with
many and all other internal organs are possible.
 It is associated with group of disorders called Leukodystrophies.
c. Werner Syndrome (Adult progeria):
 Werner syndrome is a heridatory condition associated with premature aging and
increased risk of cancer and other diseases.
 The sign of Werner syndrome is usually develop in teenage years.
 A person with werner syndrome doesn’t have a usual growth spurt typical of teenager
and is short on average.
 Sign of aging, including grey hair and hair loss may appear in the 20’s.

d. Progeria:
 It is an extremely rare genetic disorder that causes effective individual to undergo
advanced aging at an early age.
 The symptoms closely resemble aging and include wrinkles ,hair loss and delayed
growth.
 Affective individual have normal development Up to 18 months and suddenly stop
gaining weight and display stunted height .
 As the individual ages,progeria become more severe with an average life expectancy
of 12 years.
e. Rhothmund -Thomson syndrome:
 The common clinical findings are
 Sun sensitive rash with telangiectasia .
 Juvenile cataract.
 Saddle nose.
 hair growth problem (absent eyelashes , eyebrows and or hair).
 osteosarcoma.