1. DNA repair
by:S.Solali

3. Types of DNA Damage Deamination: (C  U and A hypoxanthine)
Depurination: purine base (A or G) lost
T-T and T-C dimers: bases become cross- linked, T-T more prominent, caused by UV light (UV-C (<280 nm) and UV-B ( nm)
Alkylation: an alkyl group (e.g., CH3) gets added to bases; chemical induced; some harmless, some cause mutations by mispairing during replication or stop polymerase altogether

5. Types of DNA Damage (cont.)
5. Oxidative damage: guanine oxidizes to 8-oxo-guanine, also cause SS and DS breaks, very important for organelles
6. Replication errors: wrong nucleotide (or modified nt) inserted
7. Double-strand breaks (DSB): induced by ionizing radiation, transposons, topoisomerases, homing endonucleases, mechanical stress on chromosomes, or a single-strand nick in a single-stranded region (e.g., during replication and transcription)

6. IMPORTANCE OF DNA REPAIR
Hoeijmakers
, 2001

7. DNA Repair
DNA damage may arise: (i) spontaneously, (ii) environmental exposure to mutagens, or (iii) cellular metabolism.
DNA damage may be classified as: (I) strand breaks, (ii) base loss (AP site), (iii) base damages, (iv) adducts, (v) cross-links, (vi) sugar damages, (vii) DNA-protein cross links.
DNA damage, if not repaired, may affect replication and transcription, leading to mutation or cell death.

10. Methyl-directed mismatch repair
If any mismatch escapes the proof reading mechanisms it will cause distortion of the helix.
This can be detected and repaired but it is important that the repair enzyme can distinguish the new strand from the old.
This is possible in E. coli because there is an enzyme which methylates the A in a sequence GATC. This methylation does not occur immediately after synthesis and until it does the two strands are distinguishable.

11. Mismatch repair
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.

12. Mismatch Repair in E.coli
MutS is responsible for initiation of E. coli mismatch repair.
95 kDa polypeptide, which exists as an equilibrium mixture of dimers and tetramers
recognizes mismatched base pairs.
MutL, a 68 kDa polypeptide that is dimeric in solution, is recruited to the heteroduplex in a MutS- and ATP-dependent fashion.
The MutL‚ MutS‚heteroduplex complex is believed to be a key intermediate in the initiation of mismatch repair

13. Methyl Directed MisMatch repair in E. coli

14. Methylataion and Mismatch Repair

15. Model for Mismatch Repair

17. Excision Repair • Conserved throughout evolution, found in
all prokaryotic and eukaryotic organisms
• Three step process:
– 1. Error is recognized and enzymatically clipped out
by a nuclease that cleaves the phosphodiester bonds
(uvr gene products operate at this step)
– 2. DNA Polymerase I fills in the gap by inserting the
appropriate nucleotides
– 3. DNA Ligase seals the gap

18. 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

19. Base excision Repair For correction of specific Chemical Damage in DNA
Uracil
Hypoxanthine
3-m Adenine
Urea
Formamidopyrimidine
5,6 Hydrated Thymine

20. Base excision repair. Consist of DNA glycosylases and AP endonuclease
The DNA glycosylases are specific
Uracil glycosylase
Hypoxanthine DNA glycosylase
Etc…

21. Mechanism 1.DNA glycosylase recognizes Specific Damaged base
2. Cleaves glycosl bond to remove
Base
3. AP endonuclease cleaves
Backbone
4. DNA Pol removes abasic site
5. Replacement of Base

22. Base Excision Repair (BER)
Deaminated C
Base Excision Repair (BER)
Variety of DNA glycosylases, for different types of damaged bases.
AP endonuclease recognizes sites with a missing base; cleaves sugar-phosphate backbone.
Deoxyribose phosphodiesterase removes the sugar-phosphate lacking the base.
DNA glycosylase cuts the bond between base and sugar. AP refers to Apurinic or Apyrimidinic nuclease.
Fig. 6.15

23. 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.
Ludovic C. J. Gillet and Orlando D. Scharer Molecular Mechanisms of Mammalian Global Genome Nucleotide Excision Repair Chem. Rev. 2006, 106,

24. Excision repair
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 damged strand containing the pyrimidine dimers.
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 then repaired by DNA pol I and DNA ligase.
Is error-free.

25. Excision Repair in E.coli
Damage recognised
by UvrABC, nicks
made on both sides of
dimer 5’ 3’ 3’ 5’ T T 5’ 3’ 3’ 5’ T T
Dimer removed by UvrD, a helicase T 3’ 5’ 5’ 3’
Gap filled by DNA pol I and the nick sealed by DNA ligase 3’ 5’ 5’ 3’

26. Nucleotide-Excision Repair in E. coli and Humans

27. Excision repair The UvrABC complex is referred to as an exinuclease.
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.
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)

28. Nucleotide Excision Repair
Defects cause
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, especiallyin regions exposed to sunlight such as hands, face, neck
Cockayne Syndrome
Trichothiodystrophy

29. Nucleotide Excision Repair
Defects cause
Cockayne Syndrome
A second disorder with UV sensitivity was reported by Edward Alfred Cockayne in 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

30. Nucleotide Excision Repair
Defects cause
Trichothiodystrophy
A third genetic disease characterized by UV sensitivity, trichothiodystrophy (TTD, literally: “sulfur-deficient brittle hair”), was reported by Price in 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”.

31. Photoactivation Repair in E. coli
• Exposing UV treated cells to blue light
results in a reversal of the thymine dimer
formation
• Enzyme, photoactivation repair enzyme
(PRE) absorbs a photon of light (from blue
light) and is able to cleave the bond
forming the thymine dimer.
• Once bond is cleaved, DNA is back to
normal

32. Direct Repair: Photoreactivation by photolyase

33. Alkylation of DNA by alkylating agents

34. O6-methyl G, if not repaired, may produce a mutation

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

36. Direct repair of alkylated bases by AlkB.

38. Pairing of homologous chromosomes and crossing-over in meiosis.

39. Helicase and nuclease activities of the RecBCD

40. Helicase and nuclease activities of the RecBCD

41. The RecBCD pathway of recombination

42. Recombination during meiosisis initiated by double-strand breaks.

43. RuvA and RuvB DNA helicase that catalyzes branch migration
RuvA tetramer binds to HJ (each DNA helix between subunits)
RuvB is a hexamer ring, has helicase & ATPase activity
2 copies of ruvB bind at the HJ (to ruvA and 2 of the DNA helices)
Branch migration is in the direction of recA mediated strand-exchange

44. The RuvA protein binds and forces the holiday junction into a flat planar structure.
The number of RuvA subunits in this figure is not clear. Should be 4 or 8 (see fig in Weaver).
When helicases unwind DNA only one strand of the double-helix passes through the center.
The helicase on the right is actually unwinding both the all yellow and all-blue helices, as is the helicase on the left.
Having the unwound DNA strands (2 of them) pass through the center of the helicase also promotes annealing of the heteroduplex.

45. RuvC bound to Holliday junction
The formation of the dimer in this way positions the active sites so they can cut the
Fig a

46. Models for recombinational DNA repair

47. Models for recombinational DNA repair of
stalled replication fork

48. DNA non-homologous end-joining (NHEJ)
Predominant mechanism for DSB repair in mammals.
Also exists in single-celled eukaryotes, e.g. Saccharomyces cerevisiae
Particularly important in G0/G1

49. Homologous recombination Non-homologous end-joining
DSB
DSB
Rad50, Mre11, Xrs2 complex
Resection
DNA-PKcs
Ku70, Ku80
Rad52
Rad50, Mre11, Xrs2 complex
Strand invasion
“Cleaning up” of ends
Rad51; BRCA2
DNA synthesis    
XRCC4/Ligase IV
Ligation
Ligation, branch migration, Holliday junction resolution

50. DNA-dependent protein kinase (DNA-PK)
ACTIVE
INACTIVE
KINASE

51. DNA-PK has three subunits7 8 X P K u 7 D N A - P K c s K u 8 6 9 k D a D N A - P K c s 8 3 k D a A T P A D P 4 7 k D a I N A C T I V E A C T I V E
Target sites: Ser/Thr-Gln

52. DNA-PK has three subunits7 8 X P K u 7 D N A - P K c s K u 8 6 9 k D a D N A - P K c s 8 3 k D a A T P A D P 4 7 k D a I N A C T I V E A C T I V E
… and is activated by DNA DSBs!

53. Multiple potential roles for Ku/DNA-PKcs in NHEJ

54. Model for nonhomologous end-joining
Fig
Model for nonhomologous end-joining

55. End-joining repair of nonhomologous DNA

56. 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.

57. The Error-Prone (SOS) Repair Mechanism
The error-prone repair mechanism involves DNA pol. III and 2 other gene products encoded by umuCD.
The UmuCD proteins are produced in times of dire emergency and instruct DNA pol. III to insert any bases opposite the tymine dimers, as the DNA damage would otherwise be lethal.
The risk of several mutations is worth the risk as measured against threat of death.
How is this SOS repair activated?

58. The SOS response
In response to extensive genetic damage there is a regulatory system that co-ordinates the bacterial cell response. This results in the increased expression of >30 genes, involved in DNA repair, these include:
recA - activator of SOS response, recombination
sfiA (sulA) - a cell division inhibitor (repair before replication)
umuC, D - an error prone bypass of thymine dimers
(loss of fidelity in DNA replication)
uvrA,B,C,D - excision repair
The SOS response is regulated by two key genes:
recA & lexA

59. SOS LexA normally represses about 18 genes
SOS regulon includes lexA (autoregulation), recA, uvrA, uvrB, uvrC, umuDC, sulA, sulB, and ssb
sulA and sulB, activated by SOS system, inhibit cell division in order to increase amount of time cell has to repair damage before replication.
Each gene has SOS box in promoter. LexA binds SOS box to repress expression. However, LexA catalyses its own breakdown when RecA is stimulated by ssDNA.

60. SOS
SOS repair is error-prone. This is why UV is a mutagen. May be due to RecA binding ssDNA in lesions, which could then bind to DNA Pol III complex passing through this area of the DNA and inhibit 3'>5' exonuclease (proofreading) ability. This makes replication faster but also results in more mutations.
This affect on proofreading seems to involve UmuD'-UmuC complex as well. RecA facilitates proteolytic cleavage of UmuD to form UmuD'. The UmuD'-UmuC complex may bind to the RecA-Pol III complex and promote error-prone replication.

61. SOS
Also allows Pol III to replicate past a T-dimer but introduces many mutations while doing so
Once damage is repaired, RecA no longer catalyzes cleavage of LexA (which is still being made), so uncleaved LexA accumulates and turns the SOS system off.