1. DNA REPAIR ANAND C.R. MSc. BIOTECHNOLOGY CUSAT

2. DNA DAMAGE

4. cause lipid peroxidation,

5. DNA damage

6. NFkappaB activation

7. TNF-alpha release in macrophages.Polycyclic Aromatic Hydrocarbons, (PAH) adhere to DNA

8. Types of DNA Damage

9. Direct DNA Damage

10. Random photons of ultraviolet (UV) light induce aberrant bonding between neighbouring pyrimidines (thymine & cytosine) bases on the same strand of DNA. The will prevent the replication machine from duplicating the DNA. The cell will die! This type of defect can be readily reversed by a process called photoreactivation. Visible light energy is used to reverse the defect (in bacteria, yeasts, protists, some plants, and some animals but NOT in humans)

12. An amino group of Cytosine is removed and the base becomes Uracil

13. An amino group of Adenine is removed and the base becomes Hypoxanthine

16. Repairing Damaged Bases Direct Reversal of Base Damage Excision Repair Base Excision Repair (BER) Nucleotide Excision Repair (NER) Mismatch Repair (MMR)

17. DNA Repair Pathways Direct Reversal The simplest of the human DNA repair pathways most energy efficient method involves the direct reversal of the highly mutagenic alkylation lesion O6-methylguanine (O6-mG) Carried out by the product of the MGMT gene O6-alkylguanine DNA alkyltransferase (AGT) (O6-methylguanine DNA methyltransferase)

18. Correction of the lesion occurs by direct transfer of the alkyl group on guanine to a cysteine residue in the active site of MGMT in a "suicide" reaction. The inactivated alkyl-MGMT protein is then degraded in an ATP-dependent ubiquitinproteolytic pathway.

19. The O6-mG adduct is generated in low levels by the reaction of cellular catabolites with the guanine residues in the DNA. A number of DNA-damaging chemotherapeutic agents attack the O6 position on guanine, forming the most potent cytotoxic DNA adducts known AGT activity correlates inversely with sensitivity to agents that form such O6-alkylguanine DNA adducts

20. Mechanism of action of AGT inhibition by O6-benzylguanine (BG). BG penetrates the active site pocket of AGT where it comes in contact with the sulfur of cysteine 145. A covalent transfer reaction inactivates the protein . Gerson S L JCO 2002;20:2388-2399

21. DNA-damaging chemotherapeutic agents Carmustine (BCNU) Temozolomide Streptozotocin Dacarbazine. carmustine Temozolomide dacarbazine Streptozotocin

22. O6 alkylation by temozolomide and carmustine (BCNU). The methylating agent temozolomide forms O6-methylguanine DNA adducts that induce cell death by invoking mismatch repair. The chloroethylating agent BCNU initially forms O6-chloroethylguanine DNA adducts that then rearrange to a 1,6-ethanoguanine cyclic intermediate followed by a crosslink with the cytosine directly on the opposite strand.

23. Base excision repair (BER) Multi-step process that corrects non-bulky damage to bases Oxidation Methylation Deamination spontaneous loss of the DNA base significant threat to genome fidelity and stability

24. BER has two subpathways: short patch: replaces the lesion with a single nucleotide long patch: replaces the lesion with approximately 2 to 10 nucleotides Both initiated by the action of a DNA glycosylase that cleaves the N-glycosidic bond between the damaged base and the sugar phosphate backbone of the DNA.

26. AP endonuclease and phosphodiesterase remove sugar phosphate.

29. upon detection a nuclease cuts the strand on both sides of the damage

30. DNA helicase removes the oligonucleotide

33. The MMR system if detects a mismatch, it assumes that the nucleotide on the already-methylated (parental) strand is the correct one and removes the nucleotide on the freshly-synthesized daughter strand.

35. Proteins involved in the DNA repairing of E. coli.

36. Recombinational Repair This type of repair is much more complicated than is excision repair, and requires many more gene products. The products of a number of these repair genes are induced by radiation damage, and therefore this type of repair requires protein synthesis before it can function. Because of its complexity, this type of repair makes mistakes

37. Postreplication Repair (recombinational DNA repair) The dots indicate lesions in the DNA. DNA synthesis proceeds up to a lesion and then skips past the lesion, leaving a gap in the daughter strand. Filling of the daughter strand gaps with DNA from parental strands by a recombinational process that requires a functional recA gene. Gaps in the parental strands are repaired by repair replication

38. Recombinational Excision Repair This process occurs in the part of the chromosome that was replicated prior to irradiation, i.e., where two sister duplexes were present before irradiation. After the excision of the lesion, the resulting gap is filled by the same recombinational process

39. The recombinational repair of excision gaps in E. coli. UV radiation-induced lesions are produced in both the replicated and unreplicated portions of the genome The gaps produced by excision in the unreplicated portion are repaired by the classical methods of nucleotide excision repair The gaps produced in the replicated portion of the chromosome are repaired by a recombinational process that requires both recA and recF (C-D)

40. Genetic Diseases Defects in the repair system lead to permanent DNA damage causing xeroderma pigmentosum and other genetic diseases.

43. Pathophysiology Defect in (NER) Seven xeroderma pigmentosum repair genes, XPA through XPG, have been identified. In addition to the defects in the repair genes, UV-B radiation also has immunosuppressive effects that may be involved in the pathogenesis of xeroderma pigmentosum

44. Midwest: 20 min sunlight would kill 50% of cells in culture dish 2%-10% UVB reaches basal layer of skin Leffell and Brash, Scientific American, July 1996

45. Xeroderma pigmentosum variant The defect in this condition is not in NER, but is instead in postreplication repair A mutation occurs in DNA polymerase έ Several immunologic abnormalities have been described in the skin of patients with xeroderma pigmentosum Clinical studies of the skin of patients indicate prominent depletion of Langerhans cells induced by UV radiation

46. Other defects in cell-mediated immunity impaired cutaneous responses to recall antigens decreased ratio of circulating T-helper cells to suppressor cells impaired lymphocyte proliferative responses to mitogen impaired production of interferon in lymphocytes reduced natural killer cell activity.

47. XP patients At high risk for skin cancer Must be protected from the sun and other sources of UV radiation With great caution in sun exposure, XP patients can live to middle age

49. Cockayne Syndrome Rare autosomal recessive, heterogeneous, multisystem disorder Named after English physician Edward Alfred Cockayne Characterized by: Dwarfism progressive pigmentary retinopathy birdlike facies photosensitivity

50. Forms of Cockayne syndrome CS Type I, the classic form characterized by normal fetal growth with the onset of abnormalities in the first two years of life. Impairment of vision, hearing, and the central and peripheral nervous system progressively degenerate until death in the first or second decade of life. CS Type II, otherwise known as connatal CS involves very little neurological development after birth. Death usually occurs by age 7. Has also been designated as COFS syndrome subdivided into several conditions (COFS type 1, 2, 3 (which is itself is associated with Xeroderma Pigmentosum) and type 4).

51. CS Type III rare and characterized by late onset milder than Type I and II. Xeroderma-pigmentosum-Cockayne syndrome (XP-CS) occurs when an individual also suffers from Xeroderma pigmentosum Some symptoms of each disease are expressed.

52. Genetics Mutations in the ERCC6 and ERCC8 genes The proteins made by these genes are involved in repairing damaged DNA via the transcription-coupled repair mechanism, particularly the DNA in active genes. If either the ERCC6 or the ERCC8 gene is altered, DNA damage is not repaired. As this damage accumulates, it can lead to malfunctioning cells or cell death.

53. Fanconi Anemia Fanconi anemia is one of the inherited anemias that causes bone marrow failure. It is a recessive disorder. There are at least 11 different mutations causing fanconi anemia. A*, B, C, D1, D2, E, F, G, I, J, and L. It is considered mainly a blood disease. Many patients eventually develop acute myelogenous leukemia at an early age. Patients are very likely to develop squamous cell carcinomas.

55. physical abnormalities

56. bone marrow failure

57. increased risk of malignancy.

58. Physical abnormalities short stature abnormalities of the thumbs, forearms, skeletal system, eyes, kidneys and urinary tract, ear, heart, gastrointestinal system, oral cavity, and central nervous system

59. Physical abnormalities contd. 3. Hearing loss 4. Hypogonadism 5. Developmental delay Progressive bone marrow failure with pancytopenia typically presents in the first decade, often initially with thrombocytopenia or leukopenia

60. Diagnosis and Treatment Patients are usually smaller than average. Blood tests may show a low WBC, RBC, and platelet count. Fatigue. Frequent infections. Frequent nosebleeds Easy bruising. Treatments include: Bone marrow transplant. Growth factors. Hematopoietic (blood-stimulating) growth factors are used to stimulate WBC production. Androgens. Male hormones often stimulate the production of RBCs and platelets.

61. Ataxia-telangiectasia Rare childhood disease that affects the brain and other parts of the body. Ataxia refers to uncoordinated movements, such as walking. Telangiectasias are enlarged blood vessels (capillaries) just below the surface of the skin. Telangiectasias appear as tiny red spider-like veins.

63. Complications

64. Health Comments

65. A knowledge of optimal intakes for vitamins and minerals that are needed to prevent DNA damage is required "Excessive genome instability, a fundamental cause of disease, is often an indication of micronutrient deficiency and is therefore preventable accurate diagnosis of genome instability using DNA damage biomarkers that are sensitive to micronutrient deficiency is technically feasible it should be possible to optimise nutritional status and verify efficacy by diagnosis of a reduction in genome damage rate after intervention"

66. z SUMMARY

67. REFERENCES http://www.rndsystems.com/mini_review_detail_objectname_MR03_DNADamageResponse.aspx http://www.ncbi.nlm.nih.gov/pubmedhealth/PMH0002369/ http://www.suite101.com/content/defects-in-dna-repair-and-resulting-diseases-a137961 Essential cell biology, 2/e ALBERTS BRAY GENES IX BENJAMIN LEWIN

68. THANK YOU