Mechanisms of DNA Damage and Repair

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

2. 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’ phosphodiesterbonds

6. “Volumes of history is written in the ancient alphabet of G and Cand 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. • Mutationis 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 BilalNaveed

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 • Ethylmethanesulfonate, 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)pyrenediolepoxide • 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

28. 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. Direct enzymatic Repair

29. Photoreactivation

30. Direct enzymatic Repair 2.Removal of methyl groups. Another example of Direct enzymatic repair. Mechanism

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 : • It repairs DNA bases damaged by:

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

37. Importance • The biological importance of NER is that defects in NER cause several human genetic disorders, including • Xerodermapigmentosum • 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 Prokaryotes NER in Eukaryotes

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.