DNA repair mechanisms

Introduction
• Living organisms are continuously exposed to a myriad of DNA
damaging agents that can impact health and modulate disease-
states.
• DNA damage is an alteration in the chemical structure of DNA,
such as a break in a strand of DNA, a base missing from the
backbone of DNA, or a chemically changed base in a DNA.
• DNA repair is a collection of process by which a cell identifies
and corrects damage to the DNA molecules.
• Cells are equipped with intricate and sophisticated systems—
DNA repair, damage tolerance, cell cycle checkpoints, and cell
death pathways—that collectively function to reduce the
deleterious consequences of DNA damage.
• DNA repair mechanisms are essential
processes that organisms use to correct
damaged or altered DNA. DNA damage can
occur due to various factors, such as exposure
to radiation, chemicals, or even errors during
DNA replication. Failure to repair DNA
damage can lead to mutations, which can
contribute to diseases like cancer.
 Sources of damage
• Endogenous damage:
• Endogenous damage such as attack by reactive oxygen species
produces from normal metabolic byproducts (spontaneous mutation),
especially the process of oxidative deamination, Alkylation.
• Depurination.
• Also includes replication errors.
• Exogenous damage:
• Exogenous damage caused by external agents such as ultraviolet
radiation, x-rays, gamma rays, and viruses.
• DNA damage Roughly around • 20,000 abasic sites • 10,000 oxidized
bases • 7,000 Alkylations • 10-1000 replication errors • 10 double
strand breaks Per day per cell
Mechanism of DNA Damage Response
• Cells respond to DNA damage by instigating robust DNA
damage response (DDR) pathways, which allow sufficient
time for specified DNA repair pathways to physically
remove the damage in a substrate-dependent manner.

• Recognition of DNA Damage: The first step in DNA repair

is the recognition of DNA damage. Cells have proteins and
enzymes that constantly scan the DNA for abnormalities or
lesions. These proteins can identify a wide range of DNA
damage, including mismatched bases, chemically modified
bases, and physical breaks in the DNA strands.
• Excision or Removal of Damaged DNA: Once DNA damage is
recognized, the damaged DNA is excised or removed. The specific
repair pathway depends on the type of damage and the context in
which it is found. For example, base excision repair (BER)
removes and replaces individual damaged bases, while nucleotide
excision repair (NER) removes a larger stretch of DNA containing
the damage.
• Repair Enzymes and Proteins: Various enzymes and proteins are
involved in DNA repair, depending on the type of damage and the
repair pathway being used. These include DNA glycosylases (for
BER), endonucleases (for NER), and DNA polymerases that
synthesize new DNA to replace the damaged strand.
• Synthesis and Ligation: In most DNA repair
pathways, after the damaged DNA is excised,
a complementary DNA strand is synthesized
and then ligated to the remaining DNA. This
ensures that the DNA strand is fully repaired
and intact.
• In addition, certain types of DNA damage activates DNA damage
tolerance pathways.
• In higher eukaryotes, for example, a well-orchestrated group of five
main translesion synthesis (TLS) polymerases—bypass the damage to
enable the continuation of replication, but with the possibility of a
concurrent introduction of an incorrect base that can be fixed into a
mutation in the subsequent round of replication.
• Under the circumstances, when the damaged DNA persists,
programmed cell death or apoptosis, a regulatory response to DNA
damage, is activated to get rid of cells with extensive genome
instability.
• Not surprisingly, in many cancers, DNA repair, DNA damage tolerance
and DDR pathways are disrupted.
 Categories of DNA repair
• Most cells posses different categories of DNA repair systems:
1. Direct Reversal repair
2. Base Excision Repair (BER)
3. Nucleotide Excision Repair (NER)
4. Mismatch repair
5. Double-strand break repair
6. Homologous Recombination (HR)
7. Non-Homologous End Joining (NHEJ)
8. Micro homology-mediated end joining
9. Single-Strand Annealing (SSA)
10. Trans lesion synthesis
Types of DNA repair mechanism
 Direct reversal repair
• Small subsets of DNA lesions—UV photolesions and alkylated bases—are
simply reversed in an error-free manner.
• Two different classes of enzymes reverse alkylated bases in humans and
mammals. First, the O6-alkylguanine- DNA alkyltransferase (AGT/MGMT)
enzyme reverses O-alkylated DNA lesions.
• For example, in the repair of O6-methylguanine lesions, the enzyme O6-
methylguanine-DNA methyltransferase (MGMT) transfers the methyl group
from the damaged base to itself, restoring the DNA.
• The second class of direct reversal enzymes, the AlkB related a-
ketoglutarate-dependent dioxygenases (AlkB), reverse N-alkylated base
adducts.
 Base excision repair ( BER)
• BER corrects those forms of oxidative, deamination, alkylation, and abasic
single base damage that are not perceived as significant distortions to the DNA
helix.
• Mainly active in the G1 phase of the cell cycle
• BER is responsible for repairing damaged or incorrect bases. It involves several
enzymes, including DNA glycosylases, Apurinic/apyrimidinic (AP)
endonuclease, and DNA polymerases, which work together to remove the
damaged base and replace it with the correct one.
• At least 11 different DNA glycosylases can recognize and excise a damaged base
from undistorted helices, as well as ones flipped out from the major groove.
• In short, the abasic site is the substrate for the AP endonuclease (APE1 in human
cells), which cleaves the phosphodiester bond 5” to the abasic site and generates
a hydroxyl residue at the 3-end while leaving a deoxyribose phosphate (dRP) at
the 5-end. This repair gap is tailored by the 5-dRP lyase activity of POL b (gap
tailoring), followed by filling the single nucleotide gap by POL b and ligation by
either LIG1 (DNA ligase 1) or a complex of LIG3 (DNA ligase 3) and XRCC1
(Xray repair cross-complementing protein 1)
 Nucleotide Excision Repair (NER)
• Nucleotide Excision Repair (NER): NER primarily repairs
bulky lesions, such as those caused by UV radiation or
chemicals. It recognizes and removes a larger segment of DNA
containing the damaged base, then synthesizes a new strand to
replace the excised segment.
• There are two major branches of NER: global genome NER
(GG-NER) and transcription-coupled NER (TCNER).
• NER deficiency results in a number of different human
syndromes: Xeroderma Pigmentosum (XP), associated with a
predisposition to skin cancers; Cockayne Syndrome (CS); rare
UV-Sensitive Syndrome (UVSS); and Cerebro-Oculo-Facio-
Skeletal syndrome
 Nucleotide excision repair (NER)

In GG-NER, the main DNA

damage sensor is the XPC
(Xeroderma Pigmentosum,
complementation group C)
protein, complexed with
RAD23B (UV excision repair
protein Radiation sensitive
23B) protein and CETN2
(Centrin 2). This complex scans
for the presence of transient
single-stranded DNA (ssDNA)
caused by disrupted base
pairing due to the lesion
 Mismatch Repair (MMR)
• Mismatch Repair (MMR): MMR is responsible for
correcting errors that occur during DNA replication. It
identifies and repairs mispaired bases that were not
corrected by the proofreading function of DNA polymerase.
• MMR is an evolutionarily conserved, post replicative repair
pathway that contributes to replication fidelity by at least
100-fold.
• Humans employ the MutSa heterodimer (MSH2/MSH6) to
recognize base mismatches and one-to-two nucleotide
Insertion/Deletion loops IDLs, and the MutSb heterodimer
(MSH2/MSH3) to recognize large IDLs
 Double-Strand Break Repair
• Double-Strand Break Repair:
• Double-strand breaks are one of the most
severe types of DNA damage. Cells have
several mechanisms to repair these breaks,
including homologous recombination and non-
homologous end joining. These mechanisms
involve using a homologous DNA template or
directly ligating the broken ends.
Double stranded break repair
 Homologous Recombination (HR)
• Homologous Recombination (HR): HR is a
high-fidelity repair mechanism used to repair
double-strand breaks in DNA. It uses an
undamaged homologous DNA strand as a
template to restore the broken DNA molecule.
This mechanism is essential for the repair of
complex DNA damage, such as double-strand
breaks.
 Non-Homologous End Joining (NHEJ)

• Non-Homologous End Joining (NHEJ):

NHEJ is another mechanism for repairing
double-strand breaks, but it is more error-
prone than HR. NHEJ rejoins the broken DNA
ends without using a template, often resulting
in the loss or addition of a few base pairs at the
repair site.
 Microhomology-Mediated End Joining
(MMEJ)
• Microhomology-Mediated End Joining
(MMEJ): MMEJ is another mechanism for
repairing double-strand breaks, involving short
stretches of microhomology between the
broken ends. It results in the deletion of
sequences between the microhomologous
regions.
 Single-Strand Annealing (SSA)
• Single-Strand Annealing (SSA): SSA is a
specialized form of DNA repair used to fix
double-strand breaks with extensive homology
at the ends. It involves the resection of
overhanging single-stranded DNA and
annealing of homologous sequences to repair
the break.
 Translesion Synthesis (TLS)
• Translesion Synthesis (TLS): TLS is a
mechanism that allows DNA replication to
continue past damaged bases. Specialized
DNA polymerases, known as translesion
polymerases, can replicate over the damaged
site, albeit with lower fidelity.
a)
• These DNA repair mechanisms are crucial for maintaining
the integrity of an organism's genetic information.
• Failure in these processes can lead to mutations, genomic
instability, and the development of diseases, including
cancer. DNA repair is critical for maintaining genomic
stability and preventing mutations that can lead to disease.
• Defects in DNA repair mechanisms can lead to a higher risk
of cancer and other genetic disorders. Understanding these
repair processes is essential for the development of therapies
for diseases associated with DNA damage.
Diseases associated with defective DNA repair system

• • Ataxia telangiectasia
• • Bloom syndrome
• • Cockayne's syndrome
• • Progeria (Hutchinson-Gilford Progeria syndrome)
• • Rothmund-Thomson syndrome
• • Trichothiodystrophy
• • Werner syndrome
• • Xeroderma pigmentosum
• • Hereditary non polyposis colon cancer.
Thank you