2. • DNA repair is a collection of processes by which a cell
identifies and corrects damage to the DNA molecules that
encode its genome.
• In human cells, both normal metabolic activities and
environmental factors such as UV light and radiation can
cause DNA damage, resulting in as many as
1 million individual molecular lesions per day.
• Many of these lesions cause structural damage to the
DNA molecule and can alter or eliminate the cell's ability
to transcribe the gene that the affected DNA encodes.
3. • As a consequence, the DNA repair process is constantly active as it responds
to damage in the DNA structure. When normal repair processes fail, and
when cellular apoptosis does not occur, irreparable DNA damage may occur,
including double-strand breaks and DNA cross-linkages.
4. • DNA damage can be subdivided into two main types:
• Endogenous damage such as attack by reactive oxygen species produced
from normal metabolic byproducts (spontaneous mutation), especially the
process of oxidative deamination
– also includes replication errors
• Exogenous damage caused by external agents such as
– ultraviolet [UV 200-400 nm] radiation from the sun
– other radiation frequencies, including x-rays and gamma rays
– hydrolysis or thermal disruption
– certain plant toxins
– human-made mutagenic chemicals, especially aromatic compounds that
act as DNA intercalating agents
6. Excision repair system:
• Excision repair deals with a variety of structural defects in the
DNA. Mismatches between the strands of DNA are one of the
major targets for the repair systems .
• In general , excision repair comprises 2 steps :
1. Incision step: In this step , the damaged structure is recognized by
an endonuclease that cleaves the DNA strand on both sides of
2. Excision step: In this step, a 5’to 3’ exonuclease removes a stretch
of the damaged strand .Alternatively , a helicase displaces the
damaged strand , which is subsequently degraded.
7. • Mismatches are usually corrected by excision repair . They are 2
types of excision repair :
• Base excision repair (BER)
• Nucleotide excision repair (NER)
8. Base excision repair
• This repair involve the direct removal of the damaged base
from DNA .
• This serves as the trigger to activate the enzymes that excise
& replace a stretch of DNA , including the damaged site .
• Enzymes that removes bases from DNA are called
• DNA glycosylases are responsible for initial recognition of
the lesion. They flip the damaged base out of the double helix
and cleave the N-glycosidic bond of the damaged base,
leaving an AP site.
9. • There are two categories of glycosylases:
• monofunctional and bifunctional.
• A wide variety of glycosylases have evolved to recognize different
• The AP endonucleases cleave an AP site to yield a 3' hydroxyl
adjacent to a 5' deoxyribose phosphate (dRP).
• AP endonucleases are divided into two families based on their
homology to bacterial AP endonuclease IV and exonuclease III.
10. DNA polymerase
• Pol β is the main human polymerase that catalyzes short-patch BER,
with pol λ able to compensate in its absence.
• In addition to polymerase activity, these enzymes have a lyase
domain that removes the 5' dRP left behind by AP endonuclease
• These polymerases perform displacing synthesis, meaning that the
downstream 5' DNA end is "displaced" to form a flap
11. Flap endonuclease
• FEN1 removes the 5' flap generated during long patch BER.
• This endonuclease shows a strong preference for a long 5' flap
adjacent to not 3' flap. In addition to its role in long-patch BER,
FEN1 cleaves flaps with a similar structure during Okazaki
fragment processing, an important step in lagging strand DNA
12. DNA ligase
• DNA ligase III catalyzes the nick-sealing step in short-patch BER
in humans. DNA ligase I ligates the break in long-patch BER.
• In humans, polynucleotide kinase-phosphatase (PNKP) promotes
formation of these ends during BER.
• This protein has a kinase domain, which phosphorylates 5' hydroxyl
ends, and a phosphatase domain, which removes phosphates from 3'
ends. Together, these activities ready single-strand breaks with
damaged termini for ligation.
14. Nucleotide excision repair:
• The general principles of nucleotide excision repair is similar to
• It has 2 major pathway
• Global genome repair recognizes damage anywhere in genome .
Genes called XPA to XPG are involved . The XPC protein detects the
damage and initiates the repair pathway .
• Transcription –coupled pathway is responsible for repairing lesions
occurred in the transcribed strand of active genes . In this case , the
damage is recognized by RNA polymerase 2.
15. • The 2 pathways eventually merge &
use a common set of protein to bring
about the repair .
• The strands of DNA are unwound by
approximately 24bp around the
damaged site by the helicase activity
of the transcription factor TFII, which
includes the products of 2 XP genes
XPB & XPD .
• Cleavages are on either side of the
lesion by endonuclease encoded by
XPG & XPF genes .
16. • the single - stranded stretch including the damaged bases can then
replaced by new synthesis and ligated by ligase-3 & ERCC1
• Mutations in TC-NER machinery are responsible for multiple
genetic disorders including:
• Trichothiodystrophy (TTD): some individuals are photosensitive,
• Cockayne syndrome (CS): photosensitivity, mental
retardation, progeria-like features.
17. SOS REPAIR/ ERROR–PRONE REPAIR :
• The SOS response is a global response to DNA damage in which
the cell cycle is arrested and DNA repair ,mutagenesis are induced.
• The system involves the Rec-A protein (Rad51 in eukaryotes). The
RecA protein, stimulated by single-stranded DNA, is involved in
the inactivation of the Lex-A repressor thereby inducing the
• It is an error-prone repair system that is attributed to mutagenesis.
• The SOS response was discovered and named by Miroslav
Radman in 1975.
18. • The damaged DNA cause RecA to trigger the response & results in
the auto cleavage of protein called LexA protein .
• RecA is activated on binding on a single-stranded DNA.
• Lex A is a repressor that participate in DNA repair . RecA forms a
filament around these ssDNA regions in an ATP-dependent
fashion, and becomes activated.
• The activated form of RecA interacts with the LexA repressor to
facilitate the LexA repressor's self-cleavage from the operator.
19. • Once the pool of LexA decreases, repression of the SOS genes
goes down according to the level of LexA affinity for the SOS
• Operators that bind LexA weakly are the first to be fully
• In this way LexA can sequentially activate different mechanisms
• Genes having a weak SOS box (such as uvrA, uvrB, and uvrD)
are fully induced in response to even weak SOS-inducing
20. • Thus the first SOS repair mechanism to be induced is nucleotide
excision repair(NER), whose aim is to fix DNA damage without
commitment to a full-fledged SOS response.
• This causes filamentation, and the induction of UmuDC-dependent
22. Photo reactivation/direct repair :
• Photolyases are DNA repair enzymes that repair damage caused by
exposure to ultraviolet light. This enzyme mechanism requires visible
light(300-600 nm), preferentially from the violet/blue end of the
spectrum, and is known as photo reactivation.
• Photolyase is a phylogenetically old enzyme which is present and
functional in many species, from the bacteria to the fungi to plants and to
• Photolyase is particularly important in repairing UV induced damage in
• The photolyase mechanism is no longer working in humans and other
placental mammals who instead rely on the less efficient nucleotide
excision repair mechanism
23. • Photolyases bind complementary DNA strands and break certain
types of pyrimidine dimers that arise when a pair
of thymine or cytosine bases on the same strand of DNA
become covalently linked.
• These dimers result in a 'bulge' of the DNA structure, referred to as
• Photolyases have a high affinity for these lesions and reversibly
bind and convert them back to the original bases.