all types of dna repair mechanisms

1. DNA REPAIR
Dr.Khushbu Soni
3rd Year Resident
Dept. of Biochemistry

2.  DNA damage, if not repaired, may affect
replication and transcription, leading to
mutation or cell death
 DNA repair is a collection of processes by
which a cell identifies and corrects damage
to the DNA molecules that encode its
genome.
What is DNA repair?

3. Efficient DNA replication
DNA replication is highly efficient process
Replication error rate is only 1 defect in 10 8 nucleotides
DNA repair rate is 99%
Unrepaired error rate is only 1 in 10 10
nucleotides
Human genome=6.4x10 9 base pairs

4. DNA
damage
Activation of cell cycle check
points
DNA repair
SOS
Response (synthesis of repair enzymes
Trans lesion synthesis/ bypas-Photoreactivation
-Demethylation
MMR BER NER HR NHEJ
FAILURE
Senescence
Apoptosis Mutation
If excess damage
If excess damage
No effect
Cancer
Death

5. TYPE OF DAMAGE DAMAGE MECH. REPAIR
MECH.
Single Base Alteration Depurination BER
Deamination BER
Alkylation BER
Base Analogue Incorporation BER
Mismatch Base MMR
Double Base Alteration Pyrimidine dimer NER
Purine dimer NER
Chain Break Single stranded break HR,NHEJ
Double stranded break HR,NHEJ
Cross linking Between DNA-DNA HR
Between DNA-Protein ?
Polymerase slippage Replication error in
microsettelite
MMR & NER

6. Cell cycle check point

7. DNA
damage
Activation of cell cycle check
points
DNA repair
SOS
Response (synthesis of repair enzymes
Trans lesion synthesis/ bypas-Photoreactivation
-Demethylation
MMR BER NER HR NHEJ
FAILURE
Senescence
Apoptosis Mutation
If excess damage
If excess damage
No effect
Cancer
Death

8. SOS response
 SOS repair occurs when cells are
overwhelmed by UV damage - this allows
the cell to survive but at the cost of
mutagenesis.
 SOS response only triggered when other
repair systems are overwhelmed by amount
of damage so that unrepaired DNA
accumulates in the cell.

11. DNA
damage
Activation of cell cycle check
points
DNA repair
SOS
Response (synthesis of repair enzymes
Trans lesion synthesis/ bypas-Photoreactivation
-Demethylation
MMR BER NER HR NHEJ
FAILURE
Senescence
Apoptosis Mutation
If excess damage
If excess damage
No effect
Cancer
Death

12. Direct reversal of DNA damage
Photoreactivation (the enzyme DNA photolyase
captures energy from light )
It is found in plants and some prokaryotes

13. Methylation of DNA by alkylating agents

14. Direct Repair: Reversal of O6 methyl G to G by
methyltransferase

15. DNA
damage
Activation of cell cycle check
points
DNA repair
SOS
Response (synthesis of repair enzymes
Trans lesion synthesis/ bypas-Photoreactivation
-Demethylation
MMR BER NER HR NHEJ
FAILURE
Senescence
Apoptosis Mutation
If excess damage
If excess damage
No effect
Cancer
Death

16. Mismatch repair(MMR)
• MMR system is an excision/resynthesis system that
can be divided into 4 phases:
• (i) recognition of a mismatch by MutS proteins
• (ii) recruitment of repair enzymes
• (iii) excision of the incorrect sequence,
• (iv) resynthesis by DNA polymerase using the
parental strand as a template.

17. When MMR is implicated?
 When a mismatch base is
encountered
 When DNA polymerase slippage
forms loop type defect
During replication,

18. In prokaryotes like E.coli…
Mut proteins
Mut S Mut L Mut H
Scans DNA and
recognize the
mismatch base
on daughter strand
-Links Mut S and Mut H
-Activates Mut H
-Binds the complex to
hemi methylated GATC
sequence
-Helicase activity

20. Clinical importance
hereditary nonpolyposis colon cancer ( HNPCC )
Faulty mismatch repair
Mutation in gene hMSH2 and hMLH1
Defective mismatch repair mechanism
Autosomal dominant
carcinoma

22. In eukaryotes-
MutS homologs: Msh2/Msh6 (MutSα)
Msh2/Msh3 (MutSβ)
MutL homologs: MLH1 and PMS2(MutLα)
MLH1 and PMS1(MutLβ)
MLH1 and MLH3(MutLγ)

23. TYPE OF DAMAGE DAMAGE MECH. REPAIR
MECH.
Single Base Alteration Depurination BER
Deamination BER
Alkylation BER
Base Analogue Incorporation BER
Mismatch Base MMR
Double Base Alteration Pyrimidine dimer NER
Purine dimer NER
Chain Break Single stranded break HR,NHEJ
Double stranded break HR,NHEJ
Cross linking Between DNA-DNA HR
Between DNA-Protein ?
Polymerase slippage Replication error in
microsettelite
MMR & NER

24. Microsatellite instability (MSI)
It is a condition manifested by damaged DNA due to
defects in the normal DNA repair process.
Microsatellites are repeated sequences of DNA
A dinucleotide repeat of CA, is most common
DNA polymerase slips out from these sequences
while replication and forms loop
This is corrected by MMR and NER mechanism

25. Defect in MMR will result in increase or decrease length
Microsatellites
It will cause DNA mutation

27. TYPE OF DAMAGE DAMAGE MECH. REPAIR
MECH.
Single Base Alteration Depurination BER
Deamination BER
Alkylation BER
Base Analogue Incorporation BER
Mismatch Base MMR
Double Base Alteration Pyrimidine dimer NER
Purine dimer NER
Chain Break Single stranded break HR,NHEJ
Double stranded break HR,NHEJ
Cross linking Between DNA-DNA HR
Between DNA-Protein ?
Polymerase slippage Replication error in
microsettelite
MMR & NER

28. Base excision repair
 Variety of DNA glycosylases, for different types of
damaged bases.
 They scan the genome and flips out the wrong base
 AP endonuclease recognizes sites with a missing
base; cleaves sugar-phosphate backbone.
 (Deoxyribose phosphate lyase) removes the sugar-
phosphate lacking the base.

30. Nucleotide Excision Repair
Used for repair of DNA adducts
EXAMPLES: thymine-thymine dimers
Produced by chemical and UV radiation damage

31. Nucleotide Excision repair in E.coli
1.UvrA and UvrB scan DNA to identify a distortion
2. UvrA leaves the complex ,and UvrB melts DNA
locally around the distortion
3. UvrC forms a complex with UvrB and creates nicks
to the 5’ side of the lesion
4. DNA helicase UvrD releases the single stranded
fragment from the duplex, and DNA Pol I and ligase
repair and seal the gap

33. Nucleotide excision repair in eukaryotes
Two major pathways
Global genome repair Transcription coupled repair

34. Type of protein Function
DDB1 ,DDB2(XPE)
XPC
Recognize damage
XPG -- Stabilizes TFIIH
-- Endonuclease activity
XPD
(subunit of TFIIH)
Act as helicase
XPB
(subunit of TFIIH)
Act as ATPase
XPA Involved in damage
verification
XPF endonuclease

36. Clinical Importance
 Xeroderma pigmentosum is an autosomal recessive
genetic disease .
 The clinical syndrome include marked sensitivity to
sunlight ( UV rays ) with subsequent formation of
multiple skin cancers & premature death .
 The risk of developing skin cancer is increased 1000
to 2000 fold.
 Cells cultured from patients with xeroderma
pigmentosum exhibit low activity for the nucleotide
excision repair process

37. TYPE OF DAMAGE DAMAGE MECH. REPAIR
MECH.
Single Base Alteration Depurination BER
Deamination BER
Alkylation BER
Base Analogue Incorporation BER
Mismatch Base MMR
Double Base Alteration Pyrimidine dimer NER
Purine dimer NER
Chain Break Single stranded break HR,NHEJ
Double stranded break HR,NHEJ
Cross linking Between DNA-DNA HR
Between DNA-Protein ?
Polymerase slippage Replication error in
microsettelite
MMR & NER

38. Double-strand break repair
NO TEMPLATE FOR REPAIR!!

39. End-joining repair of nonhomologous DNA

40.  Two proteins are involved in the non homologous
rejoining of a DNA break .
 Ku protein, a hetero dimer with two subunits
bind to free DNA ends & has latent ATP
dependent helicase activity .
 The DNA bound Ku hetero dimer recruits an unusual
DNA dependent Protein kinase
( DNA – PK )

41.  DNA – PK has a binding site for DNA free ends
 It allows the approximation of the 2 separated ends .
 The free end DNA/Ku/DNA – PK complex activates the
kinase activity in the later.
 DNA – PK reciprocally phosphorylates Ku .

42.  DNA – PK then dissociates from the DNA & Ku,
resulting in activation of the Ku helicase.
 This results in unwinding of the 2 ends.
 The unwound approximated DNA forms base pairs.
 The extra nucleotide tails are removed by an
exonuclease & the gaps are filled and closed by DNA
ligase .

43. Homologous recombination
Double strand break
MRN complex binds to DNA on either side of the break
Various proteins will trim back 5’ ends
Production of 3’ overhangs on ssDNA
RPA will cover ssDNA & prevents its own winding
Rad 51 produce nucleoprotein on ss DNA

44. Strand invasion in identical duplex
D-loop formation between 3’ overhang and
homologous chromosome
DNA polymerase will extend it
Holiday junction formation
DSBR pathway SDSA pathway
Cross over product Non cross over product

48. DNA
damage
Activation of cell cycle check
points
DNA repair
SOS
Response (synthesis of repair enzymes
Trans lesion synthesis/ bypas-Photoreactivation
-Demethylation
MMR BER NER HR NHEJ
FAILURE
Senescence
Apoptosis Mutation
If excess damage
If excess damage
No effect
Cancer
Death

49. Trans lesion DNA synthesis
 Occurs when the above repairs are not
efficient enough
 Prevents cell from having un-replicated
chromosome at the cost of some point
mutation
 enables replication to proceed across
DNA damage