DNA repair mechanisms

1. K. Narayanapura, Kothanur (PO), Bengaluru 560077
www.kristujayanti.edu.in
DNA repair mechanism
Dr. Manikandan Kathirvel
Assistant Professor,
Department of Life Sciences,
Kristu Jayanti College (Autonomous),
Bengaluru

2. Repair mechanism of mutation
 Repair system play a significant role in mutation process.
 As a result of repair, potentially lethal changes in DNA may
be eliminated.
 If the repair system function in error free manner, potentially
mutagenic lesions are eliminated before they can be
converted into final mutation.
Following are the repair mechanism
1. Photoreactivation
2. Excision repair system- Base excision and Nucleotide Excision repair
3. SOS repair
4. Mismatch repair
5. Post replicative recombination repair
6. Adaptive repair

3. Types of mechanisms
• Photoreactivation (also called light repair) - photolyase enzyme is activated
by UV light (320-370 nm) and splits abnormal base dimers apart.
• Base excision repair and Nucleotide excision repair (NER) - Damaged bases
or the regions of DNA unwind and are removed by specialized proteins;
new DNA is synthesized by DNA polymerase.
• Methyl-directed mismatch repair - removes mismatched base regions not
corrected by DNA polymerase proofreading. Sites targeted for repair are
indicated in E. coli by the addition of a methyl (CH3) group at a GATC
sequence.
• SOS Repair mechanism
• Demethylating DNA repair enzymes - repair DNAs damaged by alkylation.

5. 1. Photo reactivation
i. Ultraviolet light is a physical mutagen and can induce
mutation. Ultra violet radiation (254 nm) causes formation
of pyrimidine dimers (cyclobutane ring), when two
pyrimidine bases occurs together in single strand of DNA.
ii. Thymine dimer is most common one but cytosine dimer as
well as thymine-cytosine may also occurs. Thymine dimer
is a state in which two adjacent thymine molecules are
chemically joined distorting the structure of DNA, so that
impeding transcription and replication process.
iii. This pyrimidine dimer formation is lethal to the cell unless
it is corrected. A repair mechanism known as photo
reactivation can repair this mutation.
iv. When UV radiated population of bacteria is subsequently
exposed to visible light of wave length of 300-450nm, the
survival rate increases and frequency of mutation decreases.
This is due to activation of photo reactivating
enzyme photolyase encoded by the gene phr, which splits
thymine dimer.
Photoreactivation DNA repair mechanism

6. v. In the dark, the enzyme bind with thymine dimer
and in presence of visible light the enzyme split the
thymine dimers.
vi. Upto 80% of thymine dimers existing in genome
can be photoreactivated.
vii. Strains with mutations in the phr gene are
defective in light repair.
viii. Photolyase has been found in prokaryotes and
in simple eukaryotes, but not in humans.

7. 2. Excision Repair
• Two know types of excision repair:
– Base excision repair (BER)
• corrects damage to nitrogenous bases created by
the spontaneous hydrolysis of DNA bases as well as
the hydrolysis of DNA bases caused by agents that
chemically alter them.
– Nucleotide excision repair (NER)
• Repairs “bulky” lesions in DNA that alter or distort
the regular DNA double helix
• Group of genes (uvr) involved in recognizing and
clipping out the lesions in the DNA
• Repair is completed by DNA pol I and DNA ligase
Nucleotide

8. 2. Excision repair system
 The repair system remove and replace the altered bases from
damaged DNA. Excision repair system involves nucleotide
repair and base excision repair.
i. Base excision repair:
1. In this mechanism modified bases are recognized and cut
out. Mutation causes alkylation and deamination of bases
which are recognized by special DNA glycosylase enzyme.
2. Glycosylase recognizes and remove the damaged bases by
hydrolyzing the glycosidic bond (cleaving the bond
between the base and the deoxyribose sugar) and cut out the
damaged base creating AP site (apurinic or apyrimidinic
site).
3. The AP site is recognized by AP endonucleases (cleave the
sugar-phosphate backbone) which split the phosphodiester
bond on DNA strand at AP site and removes the AP sugar.
4. After the damaged nucleotide is removed, the gap is repair
by DNA polymerase I and ligated by DNA ligase.

10. ii). Nucleotide Excision repair
• In nucleotide excision repair mechanism, the defective nucleotides
are cut out and replaced. The enzymes in nucleotide excision repair
recognizes the distortion in shape of double stranded DNA
structure caused by thymine dimers or intercalating agents.
• In this form of repair, the gene products of the E.coli uvrA, uvrB
and uvrC genes form an enzyme complex that physically cuts
out (excises the damaged strand containing the pyrimidine
dimers.
• The UvrABC complex is referred to as an exinuclease. The
multi sub unit enzyme exinucleases (endonuclease and exonuclease
activity) hydrolyses two phosphodiester bond one on either side of
distortion caused by lesion creating 3’-OH group and 5’-P group.
• UvrAB proteins identify the bulky dimer lesion, UvrA protein
then leaves, and UvrC protein then binds to UvrB protein and
introduces the nicks on either side of the dimer.
• An incision is made 8 nucleotides (nt) away for the pyrimidine
dimer on the 5’ side and 4 or 5 nt on the 3’ side.
• The damaged strand is removed by uvrD, a helicase and the
resulting gap is filed by DNA polymerase-I in E. coli and DNA
polymerase E in Human and finally joined by DNA ligase.
• Is error-free.

11. 2) Nucleotide excision repair (NER) of pyrimidine dimer and other damage-induced distortions of DNA
In 1964, R.P.Boyce and P.Howard Flanders and R.Setlow
and W.Carrier- isolated UV sensitive mutants of E.coli, after
UV irradiation, showed higher rate of induced mutation in
dark.
These mutants are called as uvrA mutants. (uvr means Uv
repair).
The uvrA mutants can repair thymine dimers with the input of
light and also in the dark.
They are called as light independent repair system or
dark repair system or excision repair mechanism or
nucleotide excision repair mechanism.
The NER system in E.coli corrects not only thymine dimers,
but also other serious damage lesion or distortions of the
DNA helix.
The system involves 4 proteins: UvrA, UvrB, UvrC and UvrD-
that are encoded by uvrA, uvrB, uvrC and uvrD.

12. Nucleotide excision repair: Mechanism of Nucleotide excision repair:
i. In nucleotide excision repair mechanism, A complex of two
UvrA proteins slides along the DNA.
ii. When the complex recogonzes the pyrimidine dimer or
another serious distortion in the DNA, UvrA subunits
dissociate and a UvrC protein binds to the UvrB proteins at
the lesion.
iii. The resulting UvrBC protein bound to the DNA, makes a
cut about 4 nucleotides to the 3’ side of the damaged DNA
strand (done by UvrB) and 7 nucleotides to the 5’ side of
the lesion (done by UvrC). The multi sub unit enzyme
excinucleases (endonuclease and exonuclease activity)
hydrolyses two phosphodiester bond one on either side of
distortion caused by lesion creating 3’-OH group and 5’-
P group.
iv. UvrB is then released and UvrD binds to the 5’cut.
v. UvrD is a helicase and unwinds the region between the
cuts, releasing the short single stranded segment, thereby
the defective nucleotides are cut out and replaced.
vi. The resulting gap is filed by DNA polymerase-I in E. coli
and DNA polymerase E in Human and finally joined by
DNA ligase.

13. Nucleotide Excision Repair
• Used by the cell for bulky DNA damage
• Non specific DNA damage
– Chemical adducts
– UV photoproducts
First identified in 1964 in E.coli.
• In man, there is a similar process carried
out by 2 related enzyme complexes:
global excision repair and transcription
coupled repair.
• Several human syndromes deficient in
excision repair, Xeroderma pigmentosum,
Cockayne Syndrome, and are
characterised by extreme sensitivity to UV
light (& skin cancers).

14. If Nucleotide Excision Repair mechanism fails: it results in :
• Xeroderma Pigmentosum – 1874, when Moriz Kaposi used this term for the first time to describe the symptoms observed in
a patient.13 XP patients exhibit an extreme sensitivity to sunlight and have more than 1000-fold increased risk to develop
skin cancer, especially in regions exposed to sunlight such as hands, face, neck.
• Cockayne Syndrome: – A second disorder with UV sensitivity was reported by Edward Alfred Cockayne in 1936. Cockayne
syndrome CS) is characterized by additional symptoms such as short stature, severe neurological abnormalities caused by
dysmyelination, bird-like faces, tooth decay, and cataracts. CS patients have a mean life expectancy of 12.5 years but in
contrast to XP do not show a clear predisposition to skin cancer. CS cells are deficient in transcription-coupled NER but are
proficient in global genome NER.
• Trichothiodystrophy– A third genetic disease characterized by UV sensitivity, trichothiodystrophy (TTD, literally: “sulfur-
deficient brittle hair”), was reported by Price in 1980. In addition to symptoms shared with CS patients, TTD patients show
characteristic sulfur-deficient, brittle hair and scaling of skin. This genetic disorder is now known to correlate with mutations
in genes involved in NER (XPB, XPD, and TTDA genes). All of these genes are part of the 10-subunit transcription/repair
factor TFIIH, and TTD is likely to reflect an impairment of transcriptional transactions rather than regular defect in DNA
repair. This disorder is therefore sometimes referred as a “transcriptional syndrome”.