Molecular biology
Mechanism of repair
B. C. Muthubharathi
I M.Sc., Biotechnology

DNA Damage – A short note
 Both physical and chemical agents have the ability to alter the primary structure of DNA
 DNA susceptible to environmental damages – Exogenous, Endogenous
 Ionizing radiation
 Exposure to chemicals
 UV rays
 Cell’s metabolic products
 Absorption of thermal energy during metabolism – Tautomeric shift (rearrangement of bases)
 Leads to alteration, Mutation etc.,

Tautomeric shift
 Rearrangement of basepairs
 occur during normal metabolism
 Results in the production of structural isomers of a base
 Enhance the mispairing

But the system is lucky
 Repair mechanism
1. Automatic
2. Proteins and enzymes are available
 Process
I. Excision / removal of damaged section
II. Reconstruction of complementary removed section

Types of repair mechanism
 Excision repair mechanism
a. Nucleotide excision repair
b. Base excision repair
c. Mismatch excision repair
 Photo reactivation
 Recombinational repair
 SOS response
 Adaptive response
 Heat shock repair

Nucleotide excision repair
 Cut – patch mechanism
 Bulky lesions like thymine dimers can be removed and rearranged
 Steps
1. Recognition of bulky lesion in DNA
2. Hydrolyzing the phosphodiester bond in deoxyribose backbone in 5’ side of lesion
3. Excising the lesion ( along with a limited number of nucleotide on its 3’ end)
4. Filling the resultant gap with information given by complementary strand
5. Closing the nicked DNA to generate intact strand

Pathways
 Transcription coupled pathway
template strand is repaired
occur during transcription
lesion is signaled by RNA polymerase
 Global genomic pathway
less efficient
remaining of DNA is repaired
much slow

(1) damage recognition in the global pathway is mediated by an
XPC-containing protein complex, whereas damage recognition in
the transcription-coupled pathway is thought to be mediated by
a stalled RNA polymerase in conjunction with a CSB protein
(2) DNA strand separation (by XPB and XPD proteins, two helicase
subunits of TFIIH
(3) incision (by XPG on the 3’ side and the XPF–ERCC1 complex on the
5’ side)
(4) excision,
(5) DNA repair synthesis (by DNA polymerase )
(6) ligation (by DNA ligase I).

NER in E. Coli
 ABC excinuclease ( Uvr A, Uvr B, Uvr C)
 A complex A2B ( Uvr A, Uvr B) which scans the DNA for
any damage and binds to the lesion
 Uvr A is dissociated and leaving Uvr B – DNA complex
 Uvr C binds to Uvr B
 12-13 nucleotides are removed by Uvr D helicase
 The gap is filled by DNA polymerase I and DNA ligase

Base Excision Repair
 DNA glycosylase recognize DNA lesion and remove the N-glycosyl bond
 This cleavage creates apurine or apyrimidine site (AP site or abasic site)
 Several DNA glycosylase are found and each specific for a particular type of
DNA lesions
 DNA lesions may because of
uracil formation( deamination of cytosine)
8-oxoguanine
3-methyladenine (transfer of methyl group)

Example for BER
 REPAIR OF 8-OXOGUANINE BY DNA GLYCOSYLASE
• Inspection of G-C pair on the damaged DNA by DNA glycosylase
• When the enzyme come across the oxoGC, it inserts a specific aminoacid chain to
the DNA which creates a 180 of rotation.
• Then the base fits to the active site of enzyme and cleaved form associated sugar
• Once the altered purine or pyrimidine is removed the deoxyribose phosphate is
remaining in the site and it is excised by AP endnuclease
• Polymerization by DNA polymerase 
• Final sealing is done by DNA Ligase III

Mismatch repair
 Mismatched basepair causes a distortion in the geometry double helix
 Also called Methyl directed post replication repair system
 The mismatches are nearly always corrected to reflect the information in the
old (template) strand, so the repair system must somehow discriminate
between the template and the newly syrrthesized strand
 Steps
• Tagging the template DNA with methyl group to differentiate from newly
synthesized DNA
• Methylation is done in N6- position of adenine in 5’ GATC sequence

• The newly synthesized strand is repaired according to the information stored in the methylated
parent strand
• Mismatch repair system efficiently repair nearly 1000bp in hemi methylated strand
• MutL and MutS proteins form a complex and binds to the
mismatched basepair
• MutH protein binds to the MutL. MutH has a site specific
endonuclease activity
Proteins involved
MutL, MutS, MutH
DNA helicase II
exonuclease VII/ Rec J nuclease
exonuclease I / exonuclease X

Photoreactivation
 DNA damages like pyrimidine dimer formation due to UV radiation
 DNA photolyase enzymes are involved
 The enzyme use energy utilized from absorbed light to reverse the damage
 Photolyase has 2 cofactors – MTHF PolyGlu (N5,N10 –methenyltetrahryodfolylpolygultamate
FADH