1. DNA REPAIR

2. DNA REPAIR DNA is the only biological molecule that is repaired
DNA damage
Alteration to the chemical structure of DNA
Mutation
Change in the sequence of DNA

3. NATURE OF DNA DAMAGE Loss of bases Modification of bases
Inter/intra-strand crosslinks
DNA strand breakage (single and double strand)

4. CAUSES OF DNA DAMAGE Endogenous factors Exogenous factors Spontaneous
- Errors in proofreading
- Deamination of bases
- Depurination/Depyrimidination
Induced
- Byproducts of normal cellular processes (reactive oxygen species etc )
Exogenous factors
- UV irradiation (sunlight)
- High energy irradiation (x-rays)
- Mutagenic chemicals (Mustard gas, cigarette smoke, food additives)

5.  ERRORS IN PROOFREADING
Incorporation of the wrong base/s resulting in mismatches
Approximate error rate = 10-9

6.  DEAMINATION May be spontaneous or induced by chemicals Cytosine 
Adenine 
Guanine 
Thymine 
Uracil
Hypoxanthine
Xanthine ??

7.  DEAMINATION Deamination leads to unusual base pairing in DNA
- Uracil pairs with adenine
- Hypoxanthine pairs with cytosine

8. FAILURE TO REPAIR A DEAMINATED BASE = A POINT MUTATIONC
Parental strand A G
Mutation
Deamination
New strand C T C U T C
DNA
Replication G A G A G
New strand T C
Unchanged A G
Parental strand

9.  DEPURINATION/ DEPYRIMIDINATION
Cleavage of the glycosidic bond removes bases
Abasic (Apurinic/apyrimidinic, AP sites)
~ ,000 purines lost per mammalian cell/24 hr C T A G

10. FAILURE TO REPAIR ABASIC SITES = DELETIONS
Parental strand
Unchanged A G
New strand C T A G
DNA
Replication C T
New strand
AP site
Mutation G A
Parental strand

11.  REACTIVE OXYGEN SPECIES
Generated during normal aerobic respiration
Superoxides, O2-,
Hydroxyl ions (OH.)
H2O2
Most biological damage by OH.
Guanine
8-oxodG

12.  Exogenous – UV IRRADIATION
Dimerizes adjacent thymine residues.
The dimer creates a kink in the DNA that blocks the
progression DNA polymerase

13.  HIGH-ENERGY RADIATION
X-rays and gamma rays may directly break DNA strands
and/or generate reactive oxygen species

14.  Exogenous – CHEMICALS
Alkylating agents (e.g., mustard gas)
Add CH3/CH2CH3 groups to N and O groups of bases.
O6 of guanine particularly susceptible.
6-ethyl guanine acts as an analogue of adenine and pairs with thymine.
Polycyclic Hydrocarbons (cigarette smoke, exhaust fumes etc)

15.  Exogenous – CHEMICALS
Food Additives
Nitrates and Nitrites
Metabolized to Nitronium ion/Nitrous acid
Chemotherapeutic drugs
Base Analogues (e.g. 5-bromouracil, 5BU)
Intercalating Agents
Acridine dyes (e.g., proflavin)
Interfere with DNA replication

16. THE CELL CYCLE

17. ☺ METHODS OF REPAIR ☺ Excision repair - Base excision
- Nucleotide excision
☺ Mismatch repair
☺ Recombination repair

18. EXCISION REPAIR Recognition of damage Removal of damage
Resynthesis of gap
Ligation

19. EXCISION REPAIR Two types of excision repairs Base Excision Repair
Repair of methylated, deaminated, oxidized bases and AP sites.
Nucleotide Excision Repair
Repair of large adducts or distortion in the double helical structure of DNA (pyrimidine dimers, benzo(a)pyrene)

20. BASE EXCISION REPAIR Glycosylase AP Lyase AP endonuclease
DNA polymerase
DNA ligase

21. NUCLEOTIDE EXCISION REPAIR
Thymine dimer
urvAB excinuclease B A
urvC excinuclease A A B
DNA polymerase
DNA ligase
No thymine dimer

22. NER ASSOCIATED DISEASES
Xeroderma pigmentosum
Cockayne Syndrome
PIBIDS (photosensitivity, ichthyosis, brittle hair, impaired intelligence, decreased fertility, short stature)
- Characterized by an increased sensitivity to sunlight

23. Vignette 12
A 3-year-old boy, was referred to the dermatology clinic for evaluation of severe sun sensitivity and freckling.
On physical examination, he was photophobic and had conjunctivitis and prominent freckled hyperpigmentation in sun-exposed areas; his development and physical examination were otherwise normal.
The parents of the child revealed that they were first cousins; no one else in the family was similarly affected.
The dermatologist explained that the boy had classic features of xeroderma pigmentosum (XP), that is, "parchment-like pigmented skin".
To confirm the diagnosis, he had a skin biopsy to evaluate DNA repair and ultraviolet (UV) radiation sensitivity in his skin fibroblasts.
The results of this testing confirmed the diagnosis of XP. Despite appropriate preventive measures, the boy developed metastatic melanoma at 15 years of age and died 2 years later.
His parents had two other children; neither was affected with XP.

24. XERODERMA PIGMENTOSUM
Can be caused by defects in any one of seven different NER genes
Predisposition to skin cancer
Pigmentation abnormalities
Premalignant lesions
Degeneration of the
nervous system

25. ☺ EXCISION REPAIR Base excision repair Nucleotide excision repair
Repair of modified bases
Glycosylase removes base, leaves backbone intact
AP endonuclease cut backbone, AP lyase removes sugar
Nucleotide excision repair
Repair of adducts and large distortions in DNA double helix
Double excision removes damage as an oligonucleotide (12-13 nt in E. Coli, nt in humans)
DNA polymerase fills gap
DNA ligase seals nick

26. The forms of DNA damage mentioned so far, the repair machinery can easily tell which base(s) have been damaged.
Cytosine deamination gives rise to Uracil, which shouldn’t be present in DNA
Similarly bases modified by adducts are recognizable
as wrong.
What happens when the cell can’t easily distinguish which base is wrong.

27. ☺ Mismatch repair Repair of replication (proofreading) errors
Recognition of bases that do not form normal Watson-Crick pairs

28. ☺ Mismatch repair
How do the repair enzymes recognize which strand to fix???
CH3 ?
CH3 A G A T C T C T T C G A T C x T C T A G A G C A G C T A G ?

29. ☺ Mismatch repair MutS/MutL MutH DNA Polymerase DNA Ligase CH3 CH3 CH3

30. HEREDITARY NONPOLYPOSIS COLORECTAL CANCER (HNPCC)
Lynch syndrome
Accounts for 2 -10% of all colon cancers
Caused by defects in mismatch repair genes MSH2, MSH6, MLH1, PMS1 or PMS2

31. DNA STRAND BREAKS

32. DNA STRAND BREAKS
naturally occurring double-strand breaks per cell per day
Two mechanisms for repair
Homologous recombination repair (HRR)
Nonhomologous recombination repair (NHRR)

33. Homologous recombination repair (HRR)

34. Nonhomologous recombination repair (NHRR)

35. HRR Vs. NHRR HRR Identical copies made
Only possible in the S and G2 phase of the cell cycle
NHRR
Small deletions occur
Any time in the cell cycle

36. Defects in DNA-repair systems associated with certain cancers

37. The End!