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  1. 1. DNA RepairProfessor Wan Zurinah Wan Ngah
  2. 2. Objectives; At the end of the lecturesthe students should be able to• describe the importance of DNA repair• Describe factors that can damage DNA• describe the types of damage that can occur• explain base and nucleotide excision repair processes• explain the consequences when the damage is not repaired
  3. 3. DNA damage occursspontaneously but can increaseby various chemical, biologicaland physical factors
  4. 4. Factors• Damage occurs spontaneously eg. 10000 depurinations/d, spontaneous deamination of C to U (100/d)• Other factors that can increase DNA damage:• Biological agents eg. viruses• Chemicals eg. mutagens, carcinogens• Physical agents eg. Radiation, UV, X-rays
  5. 5. The lesion that occurs is specificto the causative factor
  6. 6. Types of DNA damage/lesions DNA Lesions CauseLoss of base (Depurination) Acid & heatAltered base Ionizing radiation, alkylating agentsWrong base Spontaneous deamination, C to U, A to HX, 5- Methyl C to TDeletion-insertion Intercalating agents eg. Acridine dyesThymine dimers UVStrand breaks Chemicals, ionizing radiation
  7. 7. Spontaneous changes that require DNA repairSpontaneous oxidative damage( red arrows)Hydrolytic attack (blue arrows)Uncontrolled methylation by S-Adenosyl Methionine (green arrows)
  8. 8. Base Deamination in DNA Natural basesUnnatural bases for DNA
  9. 9. Most frequent changes causing serious DNAdamage in cells Unnatural base
  10. 10. UV light increases the formationof thymine dimers
  11. 11. Thymine dimersare cyclobutyldimer
  12. 12. Carcinogens cause damage byaltering bases and DNA structure Cytotoxic drugs also act by altering bases and DNA structure
  13. 13. Example of a carcinogen from the dietand environment Polyaromatic hydrocarbon group includes benzo(a)pyrene (cigarette smoke) The epoxide can covalently bind to guanine base in DNA Direct or complete carcinogens Indirect or incomplete carcinogens
  14. 14. Also a carcinogen from our diet
  15. 15. Intercalation chemical into the DNAdouble helix Aromatic compounds intercalate in between base stacks. Results in insertion and addition of new bases
  16. 16. Improper or incomplete DNArepair can lead to diseaseCells invests heavily in repairmechanisms
  17. 17. Importance of DNA Repair• DNA damage DNA changes Mutation• Mutation somatic cells Cancer• Mutation gamete cells Genetic diseases (Inborn errors of metabolism)• Cell invests heavily in repair enzs• Def in repair enzs lead to diseases eg. Xeroderma Pigmentosum patients sensitive to UV, skin lesions---skin cancer etc• Bloom Syndrome : Helicase/Ligase def, cancer risk is high
  18. 18. Inherited Syndromes with Defects in DNA Repair NAME PHENOTYPE ENZYME OR PROCESS AFFECTEDXeroderma skin cancer, cellular UV nucleotide excision-repairpigmentosum (XP) sensitivity, neurological Groups A-G abnormalitiesAtaxia-telangiectasia Leukemia, lymphoma, ATM protein, a protein kinase(AT) cellular -ray sensitivity, activated by double-strand genome instability breaks, repair by homologous recombinationBRCA-1 & BRCA-2 breast and ovarian Tumour suppressor gene, cancer activated by double stranded DNA breaksBloom syndrome Cancer at several sites, stunted growth, accessory DNA helicase for genome instability replication/Ligase 1
  19. 19. Types of DNA Repair• Excision repair – Repair of thymine-thymine dimers – Apurinic/apyrimidinic repair – Removal of uracil• Direct repair – Dealkylation of G by G alkyltransferases• Mismatch repair• Recombination repair
  20. 20. Two types of excision repair arebase excision repair andnucleotide excision repair
  21. 21. Excision repair• Incision: Enz recognise type of damage. Enz involved depends on type of damage. – DNA N-glycosylases--deaminated base, alkylated or oxidised bases, bases with opened rings – Depurination--Apurinic-apyrimidinic (AP) endonuclease;• Excision of damaged base• Re-synthesis of new DNA• Nick is joined by ligase
  22. 22. Repair enzymes• In Prokaryotes-DNA polymerase I (& II)• In Eukaryotes-DNA polymerase• Ligase
  23. 23. Base excision repair is for smalllesions
  24. 24. Nucleotide excision repair is forlarge lesions such as thyminedimers
  25. 25. Cyclobutyl/ thymine dimer repair
  26. 26. Location on DNA/gene wheremutations can occurMutations can occur in the:• Coding regions or exons• Intron/exon (splicing) junction/site• Regulatory region
  27. 27. Class, Group & Type of MutationsMutation Group TypeSubstitution Synonymous Silent Non-synonymous Missense Nonsense Splice site PromoterDeletion Multiple of 3 Not multiple of 3 Frameshift Large deletion Partial/whole gene deletion
  28. 28. Class, Group & Types of Mutations: continuedInsertion Multiple of 3 Not multiple of 3 Frameshift Large insertion Partial/whole gene duplication Expansion of Dynamic mutation trinucleotide repeats
  29. 29. missense mutation:non-conservative substitution a mutation results a change in an amino acidwhere the new amino acids has a different property than theold amino acid. The protein with the new primary structuremay have reduced or no activity. Or qualitative changes with different characteristics but same biological activity
  30. 30. nonsense mutations:a mutation results in a new stop translation condon formed before the naturally occuring one.Translation is stopped prematurely and a shortenedprotein with no biological activity is made. mRNA transcripts degraded by nonsense mediated decay
  31. 31. frameshift mutation: a deletion or insertion of one base results in a change in thetranslational reading frame resulting in premature stop codon downstream. mRNA maybe degraded by nonsense mediated decay or a truncated protein is produced.
  32. 32. Summary• DNA damage can be increased by biological, chemicals and physical factors.• Lesions include loss of base, wrong base, altered base, thymine dimers, strand breaks or deletion-insertion.• Damage is repaired by repair enzymes• Mechanism of repair include base excision repair and nucleotide excision repair
  33. 33. DNA RepairA Muslim‟s speech is not exaggerated or affected.Adbdullah ibn Mas‟ud said:“By Him besides Whom there is no other god, I never sawanyone who was harsher on those who exaggerate in theirspeech than the Messenger of Allah swt, and I never sawanyone who was harsher on them after his death than AbuBakar, and I think that „Umar feared the most for them ofall people on earth”
  34. 34. Okazaki fragments have whichone of the following properties?A They are double strandedB They contain covalently linked RNA and DNAC They are RNA-RNA hybridsD They arise from the nicking of the sugar-phosphate backbone of the parental DNA chainE They are removed by nuclease activity Answer B
  35. 35. •Which one of the sequences listed below bestdescribes the order in which the followingenzymes participate in lagging strand DNAsynthesis in bacteria?1 DNA polymerase I 2 5’exonuclease3 DNA polymerase III 4 DNA ligase5 RNA polymeraseA 5,1,3,2,4B 3,2,1,5,4 ANSWER DC 5,3,4,2,1D 5,3,2,1,4E 3,2,5,1,4
  36. 36. Formation of thymine dimers
  37. 37. Questions• List the factors that can cause damage to DNA• How can cigarette smoke cause cancer?• List 6 types of lesions that can occur to DNA.• How can dietary contaminants cause damage to DNA?