DNA Damage and DNA Repair

1. DNA DAMAGE
AND DNA
REPAIR Dr. Sonia Angeline M
Assistant Professor
KJC

2. DNA DAMAGE
◦ DNA damage, due to environmental factors and normal metabolic processes inside the
cell, occurs at a rate of 1,000 to 1,000,000 molecular lesions per cell per day.
◦ The vast majority of DNA damage affects the primary structure of the double helix; that
is, the bases themselves are chemically modified.
◦ These modifications can, in turn, disrupt the molecules’ regular helical structure by
introducing non-native chemical bonds or bulky adducts that do not fit in the standard
double helix.
◦ 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) bulky adduct formation, (5) DNA
crosslinking, and (6) DNA strand breaks, including single and double stranded breaks

3. TYPES OF DNA DAMAGE
Oxidative Damage
◦ 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.
◦ The •OH can cause the formation of 8-oxo-7,8-dihydroguanine (8-oxoG) from guanine residues,
among other oxidative products,
◦ Guanine is the most easily oxidized of the nucleic acid bases, because it has the lowest
ionization potential among the DNA bases.
◦ The 8-oxo-dG is one of the most abundant DNA lesions, and it considered as a biomarker of
oxidative stress.
◦ An important oxidation product is 8-hydroxyguanine, which mispairs with adenine, resulting in
G:C to T:A transversions.

4. Alkylation of Bases
◦ Alkylating agents are widespread in the environment and are also produced
endogenously, as by-products of cellular metabolism.
◦ They introduce lesions into DNA or RNA bases that can be cytotoxic,
mutagenic, or neutral to the cell.
◦ Cytotoxic lesions block replication, interrupt transcription, or signal the
activation of apoptosis, whereas mutagenic ones are miscoding and cause
mutations in newly synthesized DNA.
◦ The most common type of alkylation is methylation with the major products
including N7-methylguanine (7meG), N3-methyladenine (3meA), and O6-
methylguanine (O6meG).

5. Deamination
◦ Deamination of cytosine is most frequent and important kind of hydrolytic
damage
◦ It is the removal of an amine group from a molecule
◦ The deamination of “C” to “U”
◦ In this “U” will cause “A” to be inserted opposite it and cause a C:G to T:A
transition when the DNA is replicated.
◦ Deamination converts adenine to hypoxanthine and guanine is converted in
to xanthine, which continues to pair with cytosine, though with only two
hydrogen bonds.

6. Base Loss
◦ An AP site (apurinic/apyrimidinic site), also known as an abasic site, is a location in DNA
(also in RNA but much less likely) that has neither a purine nor a pyrimidine base, either
spontaneously or due to DNA damage.
◦ It has been estimated that under physiological conditions 10,000 apurinic sites and 500
apyrimidinic may be generated in a cell daily.
Abasic Sites. Apurinic and Apyrimidimic (AP) sites occur
due to unstable hydrolysis.

7. Bulky Adduct Formation
◦ Bulky adducts are formed when certain chemicals, such as polycyclic aromatic
hydrocarbons (PAHs), covalently bind to DNA bases.
◦ These adducts create bulky modifications that stick out from the DNA and disrupt its
structure.
◦ They can interfere with DNA replication, transcription, and repair processes, potentially
leading to mutations.

8. UV radiation
◦ UV light can cause molecular crosslinks to form between two pyrimidine residues, commonly
two thymine residues, that are positioned consecutively within a strand of DNA.
◦ Two common UV products are cyclobutane pyrimidine dimers (CPDs) and 6–4 photoproducts.
◦ Uncorrected lesions can inhibit polymerases, cause misreading during transcription or
replication, or lead to arrest of replication.
◦ Pyrimidine dimers are the primary cause of melanomas in humans.

10. DNA REPAIR
◦ DNA damage is a common event that can interfere with important
cellular processes and lead to genomic defects and an increased risk of
cancer.
◦ To ensure the integrity of their genomes, cells have evolved to develop
mechanisms for DNA repair.
◦ These mechanisms help cells to cope with DNA damage.

11. Base excision repair
◦ Base excision repair is a mechanism used to detect and remove certain types of damaged
bases.
◦ A group of enzymes called glycosylases play a key role in base excision repair.
◦ Each glycosylase detects and removes a specific kind of damaged base.
◦ For example, a chemical reaction called deamination can convert a cytosine base into uracil, a
base typically found only in RNA.
◦ During DNA replication, uracil will pair with adenine rather than guanine (as it would if the base
was still cytosine), so an uncorrected cytosine-to-uracil change can lead to a mutation.
◦ To prevent such mutations, a glycosylase from the base excision repair pathway detects and
removes deaminated cytosines.
◦ Once the base has been removed, the "empty" piece of DNA backbone is also removed, and the
gap is filled and sealed by other enzymes.

13. • Base excision repair (BER) is a DNA repair
mechanism that removes and replaces damaged
bases.
• It involves the action of various DNA glycosylases
such as 8-oxoguanine DNA glycosylase (OGG1).
These enzymes recognize and remove damaged
bases.
• BER includes both short patch repair, where an abasic
site is processed and filled by specific enzymes, and
long patch repair, where gaps are tailored and DNA
synthesis occurs followed by ligation.
• One example of BER is the repair of uracil-containing
DNA. In this process, a DNA glycosylase recognizes
and removes the uracil base, creating a gap in the
DNA called AP site. T
• he gap is then cleaved by an enzyme called AP
endonuclease.
• After that, the remaining sugar is removed, and the
gap is filled using DNA polymerase and sealed with
ligase.

14. Nucleotide excision repair
◦ Nucleotide excision repair is another pathway used to remove and replace damaged bases.
◦ Nucleotide excision repair detects and corrects types of damage that distort the DNA double
helix.
◦ For instance, this pathway detects bases that have been modified with bulky chemical groups,
like the ones that get attached to your DNA when it's exposed to chemicals in cigarette smoke.
◦ Nucleotide excision repair is also used to fix some types of damage caused by UV radiation, for
instance, when you get a sunburn.
◦ UV radiation can make cytosine and thymine bases react with neighboring bases that are also
Cs or Ts, forming bonds that distort the double helix and cause errors in DNA replication.
◦ The most common type of linkage, a thymine dimer, consists of two thymine bases that react
with each other and become chemically linked.
◦

16. • Nucleotide excision repair (NER) deals with bulky adducts
and cross-linking lesions caused by UV radiation or
chemical exposure.
• NER removes a fragment of nucleotides containing the
damaged lesion and synthesizes a new DNA strand using
the undamaged strand as a template.
◦ NER consists of two pathways:
• Global Genome NER (GG-NER) repairs bulky damages
throughout the entire genome, including regions that are not
actively transcribed.
• Transcription-Coupled NER (TC-NER repairs damage that
occurs on the transcribed DNA strand.
• Mutations in NER pathway genes can lead to disorders
such as xeroderma pigmentosum (XP) and certain other
neurodegenerative conditions.

17. Mechanism of Nucleotide excision repair
◦ ABC excinuclease (composed of subunits coded
by uvrA, uvrB and uvrC genes) moves along
DNA and can detect Thymine dimers and for
excision endonuclease.
◦ UvrA and UvrB complex attach on distortion site
then UvrA will dissociates.
◦ UvrB attracts UvrC and nicks 5 nucleotides at 3’
side of DNA while 8 nucleotides nicks at 5’ side
of DNA will be produced by UvrC subunit.
◦ UvrD (DNA helicase II) removes 12
oligonucleotides.
◦ DNA polymerase I now fills in gap in 5'>3'
direction DNA ligase seals the gaps.

18. Mismatch repair
• Mismatch repair (MMR) pathway repairs base mismatches and insertion-deletion loops that
occur during replication. Most of these errors are fixed by the proofreading activity of DNA
polymerase during replication, but some may be missed and need to be corrected later.
• The MMR pathway involves three steps: recognition of mismatches, degradation of the error-
containing strand, and synthesis of the correct DNA sequence.
• First, protein complexes such as MSH2-MSH6 in the MutS protein locate the mismatch errors
and forms a complex with MutL which helps in further repair.
• MutS and MutL are important protein complexes in eukaryotes. In E. coli, another protein MutH
also has an important role in mismatch repair.
• Next, exonuclease 1 (Exo1) degrades the error-containing strand while replication protein A
(RPA) prevents further DNA degradation by binding to the exposed DNA.
• Then, DNA polymerase δ synthesizes the correct sequence. Finally, DNA ligase then seals any
remaining nicks in the repaired DNA.
• Mutations in MMR genes can lead to Lynch syndrome, a hereditary condition associated with an
increased risk of colon, ovarian, and other cancers.

20. Double-stranded break repair/
Recombination Repair
◦ Some types of environmental factors, such as high-energy radiation, can cause double-stranded
breaks in DNA (splitting a chromosome in two).
◦ Double-stranded breaks are dangerous because large segments of chromosomes, and the
hundreds of genes they contain, may be lost if the break is not repaired.
◦ Two pathways involved in the repair of double-stranded DNA breaks are the non-homologous
end joining and homologous recombination pathways.
◦ In non-homologous end joining, the two broken ends of the chromosome are simply glued
back together.
◦ This repair mechanism is “messy” and typically involves the loss, or sometimes addition, of a
few nucleotides at the cut site.
◦ So, non-homologous end joining tends to produce a mutation, but this is better than the
alternative (loss of an entire chromosome arm).
◦

21. ◦ In homologous recombination,
information from the homologous
chromosome that matches the
damaged one (or from a sister
chromatid, if the DNA has been copied)
is used to repair the break.
◦ In this process, the two homologous
chromosomes come together, and the
undamaged region of the homologue or
chromatid is used as a template to
replace the damaged region of the
broken chromosome.
◦ Homologous recombination is “cleaner”
than non-homologous end joining and
does not usually cause mutations.

23. Photoreactivation
◦ It is a enzymatic cleavage of thymine dimers activated by visible light.
◦ It is only present in prokaryotes (e.g. E.coli)
◦ Enzyme photolyase (encoded by phr gene) binds to a pyrimidine dimer.
◦ Visible light shines on cell then FADH absorbs that light and release electron.
◦ Electron interact with dimer.
◦ Then splitting of cyclobutane ring in dimer due to electron interaction.
◦ Finally, enzyme leaves the DNA and the DNA structure returned to its prior
state.

25. The SOS response
◦ SOS response was discovered and named by Miroslav Radman in 1975.
◦ A global response to DNA damage in which the cell cycle is arrested and DNA repair and
mutagenesis is induced.
◦ It’s a part of DNA repair system; synthesizes DNA repair enzymes.
• Named for standard SOS distress signals (stands for “Save Our Soul”), the term “SOS repair”
refers to a cellular response to UV damage
• AN SOS SYSTEM IS COMPOSED OF THE FOLLOWING COMPONENTS
• Repressor gene: encoded by the “LexA” gene - causes the inactivation of inducer proteins. The
repressor binds to the operator and causes inactivation or repression of the SOS system.
• Regulator gene: encoded by the “RecA” gene - to activate the repressed SOS system by inhibiting
the binding of LexA to the SOS operator.
• Inducer/structural genes: They are encoded by SOS-box genes that can activate the inducer
proteins relative to the type of DNA damage. -Includes- uvrA, umuC, umuD

27. • In normal DNA, a bacterial cell does not need DNA repair genes to be activated. Thus, there
should be some controller that must control the expression of such genes.
•LexA acts as a repressor protein by binding to a 20-bp consensus sequence , the SOS box.
•The binding will repress the activity of SOS genes.
• In damaged DNA, the inactivation of LexA repressor becomes necessary to induce the
expression of SOS genes.
•RecA acts as an activator of SOS genes in the SOS system, which causes proteolysis of the
repressor protein and allows the SOS genes expression into different DNA repairing inducer
proteins.

29. Mechanism of SOS Repair
◦ The mechanism of SOS repair is a complex cellular process mediated by the organism itself.
◦ It includes the following steps:
1.In case of excessive DNA damage, stress conditions etc., a cell responds by activating signal
or RecA protein. It floats in the vicinity of the cell in search of any damage in the DNA.
2.A RecA protein specifically binds to the single stranded DNA. On binding with the single
stranded DNA fragments, RecA forms a filament-like structure around the DNA.
3.Then, a LexA repressor comes in contact with the nucleoprotein filament assembled by the
RecA protein. When RecA interacts with the repressor protein, it gets converted to RecA
protease.
4.The formation of RecA protease causes autocatalytic proteolysis of LexA repressor protein.
Thus, a LexA protein cannot bind with the SOS operator.
5.Inactivation of LexA protein activates the inducer proteins that repair the DNA damage but
alters the DNA sequence.
6.After DNA repair, the RecA protein loses its efficiency to cause proteolysis, and the LexA
protein will again bind to the SOS operator or switch off the SOS system.

32. Post replication repair
◦ Post replication repair is the repair of damage to the DNA that takes place after replication.
◦ DNA damage prevents the normal enzymatic synthesis of DNA by the replication fork.
◦ At damaged sites in the genome, both prokaryotic and eukaryotic cells utilize a number of
postreplication repair (PRR) mechanisms to complete DNA replication.
◦ Chemically modified bases can be bypassed by either error-prone or error-free translesion
polymerases, or through genetic exchange with the sister chromatid.
◦ The replication of DNA with a broken sugar-phosphate backbone is most likely facilitated by
the homologous recombination proteins that confer resistance to ionizing radiation.
◦ The activity of PRR enzymes is regulated by the SOS response in bacteria and may be
controlled by the postreplication checkpoint response in eukaryotes.

33. ◦ Postreplication repair occurs downstream of the lesion, because replication is blocked at the actual site of damage.
◦ In order for replication to occur, short segments of DNA called Okazaki fragments are synthesized.
◦ The gap left at the damaged site is filled in through recombination repair, which uses the sequence from an undamaged
sister chromosome to repair the damaged one, or through error-prone repair, which uses the damaged strand as a
sequence template.
◦ Error-prone repair tends to be inaccurate and subject to mutation.
◦ Postreplication Repair Despite the accuracy of DNA polymerase action and continual proofreading, errors still are made
during DNA replication.
◦ Remaining mismatched bases and other errors are usually detected and repaired by the mismatch repair system in E.coli.
◦ The mismatch correction enzyme scans the newly replicated DNA for mismatched pairs and removes a stretch of newly
synthesized DNA around the mismatch.
◦ A DNA polymerase then replaces the excised nucleotides, and the resulting nick is sealed with a ligase.
◦ Post-replication repair is a type of excision repair.
◦ Successful post replication repair depends on the ability of enzymes to distinguish between old and newly replicated DNA
strands.
◦ This distinction is possible because newly replicated DNA strands lack methyl groups on their bases whereas older DNA
has methyl groups on the bases of both strands.