1. Mechanisms of DNA Damage and Repair
Principles of Molecular Biology

2. Maira Aleem Bilal Naveed Tanzeela Raza Maheen Malik Zaigham Abbas
Group Members
Maira Aleem
Bilal Naveed
Tanzeela Raza
Maheen Malik
Zaigham Abbas

3. DNA Structure DNA = Deoxyribose nucleic acid.
Four Nucleotides - Adenine, Cytosine, Thymine, or Guanine.
The amounts of A = T, G = C, and purines = pyrimidines [Chargaff’s Rule].

4. DNA Structure Double helix with antiparallel strands
Discovered in 1953 by James Watson and Francis Crick.
Bases on opposite strands are linked by hydrogen bonding: A with T, and G with C.
Nucleotides in each strand are linked by 5’-3’ phosphodiester bonds

6. “Volumes of history is written in the ancient alphabet of G and C and A and T.” Sy Montgomery

7. DNA Damage Alteration in
Chemical structure of DNA, such as a break in a strand of DNA, a base missing from the backbone of DNA or,
Chemically changed base

8. DNA Damage vs Mutations
Both are types of error in DNA.
DNA damage is an abnormal chemical structure in DNA.
Mutation is a change in the sequence of standard base pairs.

9. Continued… Body repairs the damage caused to DNA. Not 100% efficient.
2 points of error:
Replication of past damages in the template strand of DNA, or
During repair of DNA damages

10. DNA Repair Various checkpoints in the body.
Involved in protein synthesis that can only prevent the transduction of mutations to daughter cells by means of efficient DNA damage repair machinery.

11. Different ways of DNA Damage
Bilal Naveed

12. Factors mainly responsible for DNA Damage Radiation Hydrolysis Alkylation Oxidation

13. Radiation
High-powered particles, transmitted via X rays, alpha, beta, gamma rays etc.
X rays and gamma rays are electromagnetic waves like light, energy high and shorter wavelength
UV light is a radiation of intermediate energy that can damage cells (sunburn).

14. Radiation
Breaking the long string of letters and this can happen to one or both of the strands.
Cells are good in fixing one broken strand but both broken strands are really an issue for the cell.

15. Radiation
Double strand breaks doesn't get fixed, then part of DNA can be deleted, duplicated.
Any of these problem cause genetic disorders

16. Radiation
UV radiation in sunlight can damage DNA by messing up the base pairing
UV light will make two T's that are next to each other stick together making something called a dimer

17. Radiation
Some cells with lots of thymine dimers will die E.g. Skin peeled after a sunburn.

18. Hydrolysis
Deamination of cytosine is most frequent and important kind of hydrolytic damage
It is the removal of an amine group from a molecule
Deaminases enzyme
The deamination of “C” to “U”
In this “U” will cause “A” to be inserted opposite it and cause a C:G to T:A transition when the DNA is replicated.
Deamination converts adenine to hypoxanthine and guanine is converted in to xanthine, which continues to pair with cytosin, though with only two hydrogen bonds.

19. Hydrolysis

20. Hydrolysis
Depurination by spontaneous hydrolysis of the N-glycosyl linkage
In DNA it is a chemical reaction of purine in which the β-N-glycosidic bond is hydrolytically cleaved releasing a nucleic base

21. Alkylation
Alkylation is the transfer of an alkyl group from one molecule to another.
methyl or ethyl groups are transferred to reactive sites on the bases and to phosphates in DNA backbone.
It does not immediately leading to mispairing but they do make the bond between sugar and base more labile, or more apt to break.
It leaves an apurinic site, a sugar without its purine.
This obviously cannot be replicated properly
If they do, they frequently insert the wrong base across from an apurinic site, and this generates a mutation

22. Alkylation
Ethylmethane sulfonate, which transfers ethyl groups to DNA.
O6-rthylgaunine, often mispairs with thymine, resulting in the change of G:C base pair into an A:T base pair when the damaged DNA is replicated.

23. Oxidation
DNA oxidation is the process of oxidative damage on Deoxyribonucleic Acid
It occurs most readily at guanine residues due to the high oxidation potential of this base relative to cytosine, thymine, and adenine.
Reactive oxygen species (O2-, H2O2, and OH) are generated by ionizing radiation and by chemicals agents that generate free radicals.
An important oxidation product is 8-hydroxyguanine, which mispairs with adenine, resulting in G:C to T:A transversions.

24. Oxidation

25. Others ways of Damage
DNA damage may also result from exposure to polycyclic aromatic hydrocarbons (PAHs). 
PAHs are atmospheric pollutants commonly associated with oil, coal, cigarette smoke, and automobile exhaust fumes.
A common marker for DNA damage due to PAHs is Benzo(a)pyrene diol epoxide
It is found to be very reactive, and known to bind covalently to proteins, lipids, and guanine residues of DNA to produce BPDE adducts.
If left unrepaired,  may lead to permanent mutations and finally leading to tumor development.

26. DNA Repair Mechanisms
By: Tanzeela Raza

27. DNA Repair Mechanisms Direct enzymatic repair or Damage Reversal
1.Photoreactivation
2.Removal of methyl groups.
Single strand Damage Repair
1.Base Excision Repair
2.Nucleotide excision Repair
3. Mismatch Repair
Double strand Damage
1.Homologous Recombination
2.Non-Homologous end joining
3.SOS Response

28. Direct enzymatic Repair
1. Photoreactivation
It is a enzymatic cleavage of thymine dimers activated by visible light.
It is only present in prokaryotes (e.g. E.coli)
Mechanism
Enzyme photolyase (encoded by phr gene) binds to a pyrimidine dimer.
Visible light shines on cell then FADH absorbs that light and release electron.
Electron interact with dimer.
Then splitting of cyclobutane ring in dimer due to electron interaction.
Finally, enzyme leaves the DNA and the DNA structure returned to its prior state.

29. Photoreactivation

30. Direct enzymatic Repair
2.Removal of methyl groups.
Another example of Direct enzymatic repair.
Mechanism
Enzyme O6‑methylguanine methyltransferase (encoded by the ada gene) in E. coli, recognizes O6‑methylguanine in duplex DNA.
It then removes the methyl group, transferring it to an amino acid of the enzyme.
The methylated enzyme is no longer active, hence this has been referred to as a suicide mechanism for the enzyme.

31. Removal of methyl groups

32. Excision Repair A general mechanism of DNA repair.
Various enzymes are involved that can sense DNA damage
During excision repair bases and nucleotides are removed from damaged strands
Gap is then patched using complementarity with the remaining strand.
Excision repair is broadly categorized into
Base excision repair
Nucleotide excision repair
Mismatch repair

33. Base Excision Repair (BER)
Base from a nucleotide within DNA can be removed in several ways such as :
Radiation
By spontaneous Hydrolysis
By an attack of oxygen free radicals
By an enzyme i.e. DNA glycosylases
It repairs DNA bases damaged by:
Alkylation
Deamination
Oxidation
Lost Bases (abasic sites)

34. Mechanism of Base excision repair by using Enzyme
Uracil-DNA glycosylase enzyme recognizes uracil within DNA and cleavages it out at the base sugar (glycosidic) bond. The resulting site is called an AP (apurinic- apyrimidinic) site, because of lack of purine and pyrimidine.
AP endonucleases then sense the minor distortion of the DNA double helix and initiate excision of single AP nucleotide. AP endonucleases class I nick at 3' side of AP site and class II nick at 5' side of AP site.
DNA polymerase then inserts a nucleotide at the AP site .
Lyase or phosphodiesterase then removes the base free nucleotide.
DNA ligase then close the Nick.

35. b) Eukaryotic BER

36. Nucleotide excision repair (NER)
NER pathway can recognize and remove a wide variety of bulky, helix-distorting lesions from DNA.
It repairs DNA damaged which are produced by the UV component of sunlight.
While mechanistically similar to BER, the NER pathway is more complex, requiring some thirty different proteins to carry out a multi-step ‘cut-and-patch’-like mechanism.
General steps of NER pathway
DNA damage recognition
Local opening of the DNA helix around the lesion
Excision of a short single-strand segment of DNA spanning the lesion
Sequential repair synthesis and strand ligation

37. Importance
The biological importance of NER is that defects in NER cause several human genetic disorders, including
Xeroderma pigmentosum
Cockayne syndrome
Trichothiodystrophy
These all are characterized by extreme sun sensitivity.
In addition, these diseases demonstrate overlapping symptoms associated with cancer, developmental delay, immunological defects, neurodegeneration, and premature aging

38. Mechanism of Nucleotide excision repair
ABC excinuclease (composed of subunits coded by uvrA, uvrB and uvrC genes) moves along DNA and can detect Thymine dimers and for excision endonuclease.
UvrA and UvrB complex attach on distortion site then UvrA will dissociates.
UvrB attracts UvrC and nicks 5 nucleotides at 3’ side of DNA while 8 nucleotides nicks at 5’ side of DNA will be produced by UvrC subunit. 
UvrD (DNA helicase II) removes 12 oligonucleotides.
DNA polymerase I now fills in gap in 5'>3' direction
DNA ligase seals the gaps.

40. NER in Eukaryotes
NER in Prokaryotes

43. Mismatch repair (MMR)
The MMR system plays an essential role in post-replication repair of misincorporated bases that have escaped the proofreading activity of replication polymerases.
Accounts for 99% of all repairs 
The MMR pathway can be divided into three principle steps:
A recognition step where mispaired bases are recognized by MutS, MutL complexes.
An excision step where the error-containing strand is degraded resulting in a gap (MutH nicks progeny DNA strands).
A repair synthesis step, where the gap is filled by the DNA resynthesis.

46. Double Strand break, causes and repairing pathways
MAHEEN MALIK

47. What is double strand break?
The type of damage in which both strands of DNA are broken
DNA gets damaged by any source and both strands of DNA are broken

48. Sources
Sources that are responsible causing the Dsb are uv radiation, chemical agents, and ionizing radiation

49. DNA damage The genome of cell is continuously damaged.
It is inevitable because DNA damage arises as a result of normal cellular processes e.G. Ros
Damage can cause lesions that block the replication ultimately leading to double strand break

50. Mechanism
Our body has evolved check point mechanism that activate the repair pathways.
These checkpoints are proteins that search the whole genome that if there is any damage to the DNA.
When encounter the damage, then they turn on the mechanisms that are involved in repairing.

51. What if damage is not repaired?
If the damage is not repaired than it may result in cell death.
If not repaired correctly than it leads it to the genomic rearrangements found in many cancerous cells.

52. Pathways involved in DNA repair
Two types of pathways are involved in DNA repair mechanism
Homologous recombination repair pathway (HR)
Non homologous end joining (NHEJ)

55. Non homologous end joining (NHEJ)
Non-homologous end joining (NHEJ) functions in all kinds of cells, from bacteria to man, and is involved in DNA repair.
More common pathway
Occurs at all the stages of cell cycle

57. Mechanism
NHEJ is initiated by the recognition and binding of the Ku protein to the broken DNA ends.
Ku is a heterodimer of Ku70 and Ku80 that forms the DNA-binding component of DNA-dependent protein kinase (DNA-PK). 
It encircles the DNA and forms a bridge between the broken ends of DNA.
It then recruits DNA-PKcs that is the catalytic subunit of protein kinase.

58. Continue…
NHEJ requires the blunt ends for joining and these blunt ends are then created by artemis proteins that cut the sticky ends.
These blunt ends are then ligated by using ligase enzyme.
The DNA that is repaired is not exactly the same as damaged one because few nucleotides are missing.

60. SOS response
The SOS-response has been found in many bacterial species, but not in eukaryotic cells.

62. Mechanism The term SOS response refers to a set of co-regulated genes.
 SOS system consists of more than 40 genes and is regulated by the LexA repressor protein.

63. Continued…
Following a single-stranded DNA (ssDNA) or double-stranded DNA (dsDNA) break, activated RecA promoters assemble into filaments on chromosomal sites.
Interactions between activated RecA promoters and the LexA repressor induce the auto cleavage of LexA, which causes it to dissociate from the DNA, thereby relieving repression of the SOS regulon.

65. Diseases caused by DNA damage and repair
ZAIGHUM ABBAS

66. Factors that cause damage
Our body consist over 12 trillion cells and contains over 3 billion nucleotides in the DNA.
DNA is constantly challenged by natural and man-made chemicals, natural and man-made forms of radiation, and even by endogenous metabolism.

67. Diseases
DNA damage can lead to many different disease processes, including
Cancer
Aging
Neuro-degeneration
Cardiovascular disease
Tissue toxicities.

69. Cancer
Cells have acquired mutations, they become immortal and reproduce indefinitely as tumor cells.
Cancer-causing DNA mutations also include the loss of genes that act as tumor suppressors and the activation of oncogenes, which promote cancer.
The abnormal insertion of methyl groups into DNA at the regulatory portion of certain genes has been noted in many cancers.

70. Cancer
Sometimes people inherit a susceptibility to damage by certain environmental agents. For example, albinos have inherited an absence of skin and hair pigments, which cause DNA damage.
Most cancers arise from the accumulated mutations in our somatic cells caused by years of exposure to external toxins. E.g smoking

71. Aging
Accumulation of uncorrected DNA damage over years is a major cause of aging. Their reason is:
Animals with the fastest rates of DNA repair generally have the longest life spans.
Humans who have genetic diseases resulting in greater spontaneous DNA damage or inefficient DNA repair often show signs of premature aging.
Exposure to external causes of DNA damage (ultraviolet light, tobacco) decreases life span.

74. Neuro-degeneration
It is main feature of many nervous system and aging diseases such as
Hungtington disease
Alzheimer diseases
Parkinson’s disease

77. References
Asimuddin, M., & Jamil, K. (2012). Insight into the DNA repair mechanism operating during cell cycle checkpoints in eukaryotic cells. Biology and Medicine, 4(4), 147.
Boboye, B., & Alao, A. (2008). Effect of mutation on Trehalose-catabolic-enzyme synthesized by a tropical Rhizobium species F1. Research Journal of Microbiology, 3(4),
Mathews, L. A., & Cabarcas, S. M. (2013). DNA repair of cancer stem cells. E. M. Hurt (Ed.). Springer.
Stivers, J. T., & Jiang, Y. L. (2003). A mechanistic perspective on the chemistry of DNA repair glycosylases. Chemical reviews, 103(7),
Boiteux, S., & Jinks-Robertson, S. (2013). DNA repair mechanisms and the bypass of DNA damage in Saccharomyces cerevisiae. Genetics, 193(4),
Altieri, F., et al., DNA damage and repair: from molecular mechanisms to health implications.(Comprehensive Invited Review). Antioxidants & Redox Signaling, (5): p. 891(47).
de Laat, W.L., N.G.J. Jaspers, and J.H.J. Hoeijmakers, Molecular mechanism of nucleotide excision repair. Genes & Development, (7): p

78. Berndt, S.I., et al., Genetic variation in the nucleotide excision repair pathway and colorectal cancer risk. Cancer Epidemiol Biomarkers Prev, (11): p
Tubbs, J. L., Latypov, V., Kanugula, S., Butt, A., Melikishvili, M., Kraehenbuehl, R., ... & McGown, G. (2009). Flipping of alkylated DNA damage bridges base and nucleotide excision repair. Nature, 459(7248),
Youds, J. L., Barber, L. J., Ward, J. D., Collis, S. J., O'Neil, N. J., Boulton, S. J., & Rose, A. M. (2008). DOG-1 is the Caenorhabditis elegans BRIP1/FANCJ homologue and functions in interstrand cross-link repair. Molecular and cellular biology, 28(5),