DNA REPAIR

MECHANISM

ACHIEVERS ACADEMY
DNA DAMAGE
● DNA damage refers to any alteration or mutation that occurs
in the DNA sequence of an organism.
 TYPES OF DNA DAMAGE
1.Single Base Alteration
2.Two Base Alteration
3.Chain Break (Single or Double-Strand Breaks)
4.Crosslinkage
 TYPES OF DNA DAMAGE
1. Single Base Alteration
A change or damage to a single nitrogenous base in the DNA
sequence.
• Causes:
• Spontaneous deamination (e.g., cytosine to uracil)
• Oxidation (e.g., guanine to 8-oxoguanine)
• Alkylation (e.g., methylation of adenine or guanine)
• UV light or X-rays causing minor modifications
 TYPES OF DNA DAMAGE
2. Two Base Alteration
Damage that affects two adjacent bases, often disrupting base
pairing.
• Causes:
• UV radiation, leading to thymine dimers (covalent bonds
between two thymines)
• Chemical mutagens (e.g., bifunctional alkylating agents)
causing crosslinking or adducts between adjacent bases
• Ionizing radiation can also induce clustered damage to
nearby bases
 TYPES OF DNA DAMAGE
3. Chain Break (Single or Double-Strand Breaks)
The phosphate backbone of the DNA strand is broken, either
in one strand (SSB) or both (DSB).
• Causes:
• Ionizing radiation (X-rays, gamma rays)
• Reactive oxygen species (ROS)
• Certain chemicals (e.g., bleomycin)
• Mechanical stress or enzymatic cleavage (e.g., during
replication)
 TYPES OF DNA DAMAGE
4. Crosslinkage
Covalent bonding between two DNA strands (interstrand) or
within the same strand (intrastrand), or between DNA and
proteins.
• Causes:
• Chemotherapeutic agents (e.g., cisplatin)
• UV radiation
• Formaldehyde or other crosslinking chemicals
• Endogenous aldehydes
 THE DNA DAMAGE RESPONSE
• The DNA damage response (DDR) is a complex biological process that occurs
in cells in response to DNA damage.

• Significance: The DDR is a critical mechanism that helps maintain the

integrity of the genome and prevent the development of mutations and other
DNA-related disorders.

• The DDR is initiated when DNA damage is detected by specialized sensors

within the cell, which activate a cascade of signaling pathways that lead to
various cellular responses.

• NB: These responses can include Cell cycle arrest, DNA repair, and apoptosis
(programmed cell death), depending on the severity and type of DNA
 THE DNA REPAIR

• The DNA repair pathway is a critical component of the DDR, as it enables

the cell to repair damaged DNA and prevent the accumulation of mutations.

• This pathway can be grouped itwo based on the scale of DNA damage
incurred. They include the single-stranded DNA repair & double-
stranded repair.

A. Single-stranded Repair pathway

• This category of repair pathway is suitable for situations arising when there
is a damage to the nucleotide sequence occurring in one polynucleotide
strand. Examples of single-stranded repair pathway includes: Base
excision Repair, Mismatch repair & Nucleotide excision Repair.
 THE DNA REPAIR

A. Single-stranded Repair pathway

• Base excision Repair

• Mismatch repair

• Nucleotide excision Repair

Base Excision Repair (BER)
Base excision repair (BER) is a cellular mechanism for
repairing damaged or modified DNA bases. This
damage can occur as a result of biological activities leading
to modification of nucleotide bases.
● Examples of the processes includes: Hydrolysis (deamination
during nucleotide breakdown), Alkylation and Oxidation.
● BER is a multistep process that involves several different
enzymes and cofactors and is generally considered to be
one of the most important DNA repair mechanisms in cells.
● Enzymes: DNA glycosylases, AP endonuclease, DNA
polymerase 1, and DNA Ligase
Base Excision Repair (BER)
Base Excision Repair (BER)
● Step 1:
● Recognition & Removal of damaged base: This can be done by DNA
glycosylases, which are enzymes that recognize and remove damaged or
modified DNA bases. DNA glycosylases can recognize several different
types of base damage, including oxidized bases, alkylated bases, and
deaminated bases.
● The action of DNA glycosylase cleaves the glycosidic bond between the
damaged base and the sugar-phosphate backbone of the DNA molecule.
● This leaves an abasic (apurinic/apyrimidinic) site, which is a gap in the DNA
strand where the base used to be.
Base Excision Repair (BER)
● Step 2:
● Strand nicking and phosphate group removal: This is carried out by an AP
endonuclease, which recognises the abasic site and cleaves the DNA strand at the
site of the missing base. This generates a nick in the DNA strand, which exposes
the 3'-OH group of the sugar-phosphate backbone.
● The AP endonuclease equally has a lyase activity that cleaves away the exposed
phosphate group in the same reaction.
● Step 3:
● Excision Repair: This is also known as gap-filling. This is carried out by a DNA
polymerase 1, which incorporates the right nucleotide.
● The DNA polymerase extends the 3'-OH group of the existing DNA strand by adding
nucleotides in a complementary manner to the template strand.
Base Excision Repair (BER)
● Step 4:
● Gap sealing: The final step in the BER pathway is the sealing of
the nick.
● This is carried out by a DNA ligase, which catalyzes the
formation of a phosphodiester bond between the 3'-OH group
of the existing DNA strand and the 5'-phosphate group of the
newly synthesized DNA strand. This seals the nick and restores
the integrity of the DNA strand.
B. Mismatch Repair (MMR) pathway:
● The mismatch repair (MMR) is a critical DNA repair
mechanism that plays a vital role in maintaining the
stability of the genome by correcting errors that
occur during DNA replication.
● It is a complex process that involves a series of steps
that are highly regulated and tightly controlled to
ensure the accurate repair of mismatches
● Specific proteins and enzymes needed for MMR are:
Dam methylase
● MutH, MutL, MutS proteins, DNA helicase II, SSB, DNA
polymerase III Mismatches Exonuclease I, Exonuclease
VII, RecJ nuclease, Exonuclease X, and DNA ligase.
Mismatch Repair (MMR) pathway:
Step 1:
● Recognition of Mismatch: This is carried out by the MMR proteins.
● The proteins responsible for this step are MutS. It scans the newly
synthesized DNA strand for mismatches.
● When MutS recognizes a mismatch, it undergoes a conformational change,
and MutL is recruited to the site of the mismatch
Step 2:
● Assembly of MMR proteins: Once the MutS/MutL complex has recognized
the mismatch, it recruits other MMR proteins to the site of the damage.
These proteins include MutH, Exonuclease 1 (EXO1), DNA polymerase
delta, and DNA ligase.
 Mismatch Repair (MMR) pathway:
Step 3:
● Strand nicking & Excision of base: For this to occur, MutH breaks the
phosphodiester bond. MutH is a DNA endonuclease that cleaves the
unmethylated DNA strand near the mismatch. This cleavage exposes the
flapping nucleotide base, which is removed by an exonuclease.
Step 4:
● Gap filling: This stage is also called Excision repair.
● This is carried out by a DNA polymerase 1, which re-synthesises the
excised region of DNA.
● The DNA polymerase extends the 3'-OH group of the existing DNA strand
by adding nucleotides in a complementary manner to the template strand.
Mismatch Repair (MMR) pathway:
Step 5:
● Gap sealing: After the correct nucleotides have been
incorporated, the final step in the MMR pathway is the ligation
of the newly synthesised DNA strand to the existing strand.
The protein responsible for this step is DNA ligase, which seals
the nick in the DNA.
 C. Nucleotide Excision Repair (NER)
● Nucleotide Excision Repair
(NER) is a conserved DNA repair
mechanism that is responsible
for removing a wide range of
DNA lesions, such as bulky
adducts and UV-induced
dimers (photoproducts), and
other helix-distorting lesions
from the genome.
 C. Nucleotide Excision Repair (NER)
● Step 1:
● Recognition of DNA Lesion: The first step in the prokaryotic NER pathway is the
recognition of the lesion by the UvrA and UvrB proteins.
● The UvrA protein is responsible for the initial recognition of DNA damage, and it
forms a complex with UvrB. The UvrB protein then scans the DNA strand in
search of damage and recognizes the site of the lesion.
● Step 2: Assembly of Pre-incision complex:
● After the lesion is recognized, the UvrB protein recruits additional proteins to
form the pre-incision complex. This complex consists of UvrC and UvrD proteins.
● The pre-incision complex verifies the presence of DNA damage and positions
the DNA for subsequent incisions.
 C. Nucleotide Excision Repair (NER)
● Step 3:
● Unwinding of DNA & Incision: This process is initiated by the helicase activity of
UvrB. The unwinding creates a bubble structure that exposes the lesion to the
endonuclease activity of the UvrC protein, which makes an incision on the 3' side of
the lesion.
● The UvrC protein makes another incision on the 5' side of the lesion, releasing the
damaged DNA fragment.
● Step 4:
● Excision of Damaged DNA part: After the incisions, the damaged DNA fragment is
released from the DNA strand by the helicase activity of the UvrD protein. The
excision of the damaged fragment leaves a single-stranded DNA gap that needs to
be filled in by the DNA repair machinery.
 C. Nucleotide Excision Repair (NER)
● Step 5:
● DNA synthesis & Ligation: After the damaged DNA fragment is
excised, the gap is filled in by the action of DNA polymerases, which
synthesize a new strand complementary to the undamaged strand.
● The synthesis is directed by the DNA polymerase I, which also
removes the RNA primer used in the synthesis. The gap is then sealed
by the DNA ligase protein, which covalently links the newly
synthesized DNA strand to the original DNA strand, restoring the
integrity of the DNA.
Summary
• Base Excision Repair (BER) is used when there is a small, non-
helix-distorting base lesions, such as oxidative damage, alkylation,
deamination, and depurination.
• Enzymes and Proteins Responsible:
1. DNA glycosylases
2. AP endonuclease (APE1)
3. DNA polymerase β (Pol β)
4. DNA ligase
• Mechanisms (follow the enzymes):
• BER is initiated by DNA glycosylases that recognize and remove
the damaged base, creating an apurinic/apyrimidinic (AP) site.
• AP endonuclease then cuts the DNA backbone at the AP site.
• DNA polymerase β inserts the correct nucleotide.
• The DNA strand is sealed by DNA ligase III
 Summary
• Mismatch Repair (MMR) is used for correcting base mismatches that occur during DNA replication
and recombination, including small insertions and deletions. (Remember: parent strands are usually
methylated while the newly synthesized strand is not)
• Enzymes and Proteins Responsible:
1. MutS
2. MutL
3. Exonuclease 1 (EXO1) and MutH
4. DNA polymerase δ
5. DNA ligase I
• Mechanisms
• MMR is initiated by MutS recognizing the mismatch. MutL is recruited to the site to form a complex.
• The complex recruits exonuclease 1 and MutH breaks the phosphodiester bond and cleaves the
unmethylated DNA strand near the mismatch. which removes a section of the newly synthesized
strand containing the mismatch.
• DNA polymerase δ synthesizes the correct DNA segment.
• DNA ligase I seals the remaining nick.
• Nucleotide Excision Repair (NER) is used for removing bulky, helix-distorting lesions such as thymine dimers and chemically induced
adducts.
• Enzymes and Proteins Responsible
• UvrA (initial damage recognition), UvrB (Scans and recognize)
• UvrC and Uvr D (Complexes with UvrB forms a pre-incision complex verifies the damage)
• DNA polymerase δ or ε
• DNA ligase I
• Mechanism
• NER starts with damage recognition by UvrA. UvrB protein recruits additional proteins to form the pre-incision complex. This complex
consists of UvrC and UvrD proteins.
• helicase activity of UvrB in the pre-incision complex unwinds the DNA around the lesion.
• UvrC make incisions on either side of the lesion.
• The damaged oligonucleotide is removed and DNA fragment is released from the DNA strand by the helicase activity of the UvrD protein.
• DNA polymerase δ or ε fills in the gap.
• DNA ligase I seals the strand.
 THE DNA REPAIR

B. Double-stranded Repair pathway

(DSR)

DSR pathway mechanisms are used to repair

double-strand breaks (DSBs) in DNA.

This DNA damage occurs when both strands

of the DNA helix are broken, which can lead
to chromosomal rearrangements, mutations,
or cell death if not properly repaired.

• Homologous Recombination (HR)

• Non-Homologous End Joining (NHEJ)

 A. Homologous Recombination (HR)
● HR uses a homologous sequence (usually from the sister chromatid) as a
template for accurate repair.
● As a result, this repair mechanism can occur mainly in the S and G2 phases of the
cell cycle, when a sister chromatid is available.
Steps:
1. Detection and Resection of the Break
2. Formation of Single-Stranded DNA Tails
3. Strand Invasion and D-Loop Formation
4. DNA Synthesis and Second-End Capture
5. Ligation and Completion
● Accuracy: High-fidelity repair (error-free).
● Key proteins: Rad51, BRCA1, BRCA2, Mre11-Rad50-Nbs1 (MRN complex).
 Homologous Recombination (HR)
Steps:
● Step 1: The cell recognises the damage and initiates end
resection, where 5′ ends near the break are digested to produce
3′ single-stranded DNA (ssDNA) overhangs.
● Step 2: The DNA ends are now resected, and we observe long 3′
overhangs on each side of the break. This processing is
necessary to allow Rad51 to coat the ssDNA and prepare it for a
homology search.
● Step 3: One of the 3′ ssDNA tails invades a homologous
sequence on the sister chromatid (blue DNA). This process
forms a displacement loop (D-loop), where the invading strand
pairs with the complementary strand in the sister chromatid.
This sets up a template for DNA synthesis, using the intact
sister chromatid as a guide.
 Homologous Recombination (HR)

● Step 4: The 3′ end of the invading strand is

extended by DNA polymerase, synthesising
new DNA (shown in green).
● Step 5: The second broken DNA end is also
paired with the complementary region on
the sister chromatid, completing the repair
template structure.
● Step 6: The new DNA strands are ligated
(joined together) to restore the integrity of
the double helix.
 B. Non-Homologous End Joining (NHEJ)
● Directly ligates the broken DNA ends without needing a homologous
template.
● This repair mechanism can be used throughout the cell cycle, but especially
in the G1 phase.
Steps:
1. Detection and binding of DNA ends by Ku70/Ku80.
2. Recruitment of DNA-PKcs and other processing enzymes to trim or fill in
ends.
3. Ligation of ends by the DNA ligase IV/XRCC4/XLF complex.
● Accuracy: Error-prone, may cause insertions or deletions (indels).
● Key proteins: Ku70/Ku80, DNA-PKcs, Ligase IV, XRCC4.
 B. Non-Homologous End Joining (NHEJ)
● Directly ligates the broken
DNA ends without needing a
homologous template.
1. Detection of Double-Strand
Break
2. Binding of Ku70/Ku80
Heterodimer
3. Recruitment of Additional
Factors
4. End Joining by DNA Ligase IV
Diseases Associated with Defective
DNA Repair System
 Defective DNA repair mechanisms
● Xeroderma pigmentosum
● Bloom Syndrome
● Werner’s disease
● Hereditary non-polyposis colon cancer.
● Ataxia-telangiectasia
● Cockeyne’s syndrome
 1. Xeroderma pigmentosum
● Xeroderma Pigmentosum (XP) is a rare genetic disorder
characterised by an extreme sensitivity to ultraviolet
(UV) rays from sunlight.
● It results from defects in the nucleotide excision repair
(NER) pathway, which is responsible for repairing DNA
damage caused by UV light, particularly thymine dimers and
other photoproducts.
● In XP patients, NER is defective, so UV damage accumulates,
leading to mutations, genomic instability, and a greatly
increased risk of skin cancers.
● It is inherited by Autosomal recessive where a child must
inherit two defective copies of an XP gene (one from each
parent).
 1. Xeroderma pigmentosum
Symptoms and Clinical Features
● Early and severe sunburns after minimal sun exposure.
● Freckling and hyperpigmentation at an early age (usually before age 2).
● Dry, scaly skin (xeroderma).
● Eye abnormalities: photophobia, conjunctivitis, keratitis.
● Neurological problems (in some patients): cognitive decline, hearing loss, poor coordination.
● Skin cancer (basal cell carcinoma, squamous cell carcinoma, melanoma) often develops in
childhood.
Treatment and Management
● Strict avoidance of UV exposure: protective clothing, sunscreen, UV-blocking window films.
● Regular skin checks to monitor for cancer.
● Surgical removal of skin cancers when detected.
● Vitamin D supplementation (due to limited sun exposure).
● Gene therapy and DNA repair enhancement are being researched.
 2. Bloom Syndrome (BS)
● Bloom Syndrome is a rare inherited disorder that falls under
the category of chromosomal instability syndromes.
● It is marked by growth deficiency, sun-sensitive skin changes,
immune system problems, and a greatly increased risk of
various cancers from an early age.
● This condition is caused by mutations in the BLM gene,
which encodes a protein from the RecQ helicase family.
● RecQ protein helps in maintaining DNA stability by helping
unwind DNA during replication and repair.
● When BLM is defective, cells show a high rate of sister
chromatid exchange and chromosomal breakage, leading to
genomic instability.
 2. Bloom Syndrome (BS)
Symptoms
● Children with Bloom Syndrome typically present with short stature and a slender build.
● One hallmark feature is a butterfly-shaped rash across the cheeks and nose that becomes more
noticeable with sun exposure.
● Individuals often suffer from recurrent infections due to immunodeficiency and may develop diabetes.
● A very high risk of developing cancer, often multiple types, at a younger age than usual. Infertility is
also common.
Treatment and Management
● There is currently no cure for Bloom Syndrome.
● Management focuses on minimising exposure to sunlight by using protective clothing and sunscreen.
● Regular medical surveillance is essential to detect cancers early. T
● reating infections promptly, monitoring blood sugar levels, and supporting growth and nutrition are also
important aspects of care.
 3. Werner’s Disease (WS)
● Werner’s Disease, sometimes called adult progeria, is a
rare genetic condition that mimics normal ageing but
starts much earlier in life.
● Unlike Bloom Syndrome, which appears in childhood,
Werner’s Disease typically becomes apparent during
adolescence or early adulthood.
● Werner’s Disease is caused by mutations in the WRN
gene, which also encodes a RecQ helicase protein. This
helicase is important for DNA repair, replication,
recombination, and telomere maintenance.
● When the WRN protein is absent or malfunctioning, cells
age prematurely due to accumulated DNA damage and
shortened telomeres, leading to tissue deterioration.
 3. Werner’s Disease (WS)
● Symptoms
● People with Werner’s Disease appear normal until their teenage years, after which they start
showing signs of accelerated aging. These include premature graying and thinning of hair, early
onset of cataracts in both eyes, wrinkled or tight skin, osteoporosis, and type 2 diabetes.
Individuals may also experience infertility, atherosclerosis, and have a higher risk of developing
certain cancers. The aging-related symptoms progress over time, and many patients have a
shortened lifespan, often passing away in their 40s or 50s due to cancer or cardiovascular disease.
Treatment and Management
● As with Bloom Syndrome, there is no cure for Werner’s Disease. Management involves treating
individual symptoms as they arise. Cataract surgery may be necessary, and diabetes,
osteoporosis, and cardiovascular risks must be actively monitored and controlled. Physical therapy
and medications can help manage joint stiffness and bone health. Regular cancer screenings are
essential, and patients should receive comprehensive care from a multidisciplinary team. Genetic
counseling is also important for patients and their families.