MUTASI

1. MUTASI

3. Mutasi C U

4. Mutasi, mutan & mutagen • Mutasi • Perubahan basa dalam sekuen DNA (Pd umumnya • di dalam suatu gene). • Perubahan ini termasuk : substitusi basa, • adisi, penyusunan ulang dan delesi • Mutan • Organisme yang mengalami mutasi. • Mutasi tersebut tentunya terjadi dalam gena yang • menyebabkan terjadinya perbedaan yang nyata • dengan bentuk normal (Wild-Type). • Mutagen • agen fisika atau senyawa kimia yang menyebabkan mutasi.

5. Mutagenesis A spontaneous mutation occurs once in 108 cells. The mutation rate can be increased by exposing cells to mutagens, which are either chemicals or physical agents such as UV-irradiation. Both chemicals (EMS) and physical agents act by causing genetic damage that results in base changes in DNA

6. Causes of DNA Damage: Endogenous & Exogenous Radiation Ionizing radiation (, x-ray;  particle) double-strand breaks with 3’phosphates UV radiation pyrimidine dimers (thymine dimers, thymine-cytosine dimers) Oxidants Reactive oxygen species (H2O2 , O2- ), often generated by respiration 8-oxo-dG, 2-OH-dA Chemical mutagens: Alkylating agents (e.g., ethyl- and methyl-methane sulfonate: 3-Me and 3-ethyl-dA) Nucleophiles (e.g., hydroxylamine, catalyzes cytosine deamination) Cross-linkers (e.g. psoralen) Intercalators (e.g., acridine orange, ethidium bromide): cause deletions & insertions (i.e., frame-shifts) during replication Spontaneous deamination: uracil, xanthine, hypoxanthine: repaired by excision repair or by glycosylase (no xanthine-specific glycosylase) Replication errors: misincorporation resulting in mismatched base pairs

7. Mismatches  saat DNA replikasi Mutan Replikasi 5’-ATGGG-3’ 3’-TACCC-5’ 5’-ATTGG-3’ 3’-TAACC-5’ 5’-ATGGG-3’ 3’-TAACC-5’ Normal 5’-ATTGG-3’ 3’-TAACC-5’

8. Perubahan struktur  Mutasi DNA Mutan 5’-ATXGG-3’ 3’-TAGCC-5’ Kerusakan nukleotida 5’-ATXGG-3’ 3’-TAGCC-5’ 5’-ATXGG-3’ 3’-TAACC-5’ 5’-ATCGG-3’ 3’-TAGCC-5’ 5’-ATTGG-3’ 3’-TAACC-5’ Mutan Normal

9. Tipe mutasi Mutasi pada tingkat DNA 1. Point mutation a. Substitusi satu basa dengan nukleotida yang lain 2 tipe: Transisi – perubahan purine ke purine (A to G, G to A) atau pirimidin ke pirimidin (C to T, T to C) Transversi – perubahan purineke pirimidin atau sebaliknya, e.g. A to C or T, C to A or G b. Insersi atau delesi Penambahan atau pengurangan satu base-pairs.

10. Tipe mutasi Inversi Pemotongan sebagian DNA dan kemudian disisipkan pada tempat yg sama tetapi pada orientasi yg berbeda 5’-ATCAG-3’ 3’-TAGTC-5’ 5’-ATTGG-3’ 3’-TAACC-5’

11. Reversion This is the reverse processof mutation and involves a mutant regaining a wild-type phenotype, either through: 1. A back mutation (direct reverse of the mutation) 2. A reverse or suppressor mutation (not a direct reverse, but a mutation at a second site generally within the same gene which suppresses the effect of the first mutation). The mutant is described as a revertant. A practical example of reversion is the Ames test which is used to test for carcinogens.

12. What is reversion? WT, active Mutate - to + Mutant, inactive + + - + Mutate + to - + - - + WT, active Same site revertant WT ?, active different site revertant

14. Mutasi pada aras gena • Silent mutation • Perubahan basa tidak meyebabkan perubahan pada kodon • 5’ ATGGGA GCT CTA TTA ACC TAA 3’ • met gly ala leu leu thr stop • 5’ ATGGGA GCT CTA TTG ACC TAA 3’ • met gly ala leu leu thr stop Silent mutation ( & transition)

15. Mutasi pada aras gena • Missense mutation • Perubahan basa  Perubahan kodon • 5’ ATGGGA GCT CTA TTA ACC TAA 3’ • met gly ala leu leu thr stop • 5’ ATGGGA GCT CTA TTT ACC TAA 3’ • met gly ala leu phe thr stop Missense mutation ( & transversion)

16. Mutasi pada aras gena 3. Nonsense mutation Perubahan basa  menjadi stop kodon 5’ ATGGGA GCT CTA TTA ACC TAA 3’ met gly ala leu leu thr stop 5’ ATGGGA GCT CTA TGA ACC TAA 3’ met gly ala leu stop Nonsense mutation ( & transversion)

17. Mutasi pada aras gena 4.Frameshift mutation Hilangnya satu basa  perubahan pada pembacaan 5’ ATGGGA GCT CTA TTA ACC TAA 3’ met gly ala leu leu thr stop 5’ ATGGGG AGC TCT ATTAAC CTA A 3’ met gly ser ser ile asn leu Frameshift mutation

18. Apa akibatnya jika • Delesi 1, 2 atau 3 basa • Adisi 1, 2 atau tiga basa • Substitusi 1, 2 atau 3 basa • Baik berurutan maupun tidak berurutan

22. Spontaneous point mutation

23. DNA T RNA UUntuk menaikkan kebenaran pesan genetik • URACIL DNA GLYCOSYLASE • ENZIM MENGHIDROLISA IKATAN GLIKOSIDIK ANTARA U DAN GULA

26. Direct Damage Reversal: correction of pyrimidine dimers UV irradiation (200-300 nm) causes formation of pyrimidine dimers in which consecutive pyrimidine residues are linked by a cyclobutane ring: Pyrimidine dimer formation can be directly reversed by Photolyase which absorbs photons (300-500 nm) and catalyzes reversal of the photo-cyclization reaction. cyclobutane ring in thymine dimer

27. Nucleotide excision repair in Eukaryotes Multi protein complex (> 16 proteins) Deficiency of NER are manifested by diseases: Xeroderma pegmentosum - inability of skin cells to repair UV-damadge Individuals with this disease are extremely sensitive to UV-light, higher level of skin cancer Cockayne syndrome – defects in same genes as Xeroderma pegmentosum + two additional genes. Sensitivity to UV-ligh, neuron denyelination, normal level of skin cancer.

28. hu (300 -500 nm) Pyrimidine dimer chromophore FADH- Pyrimidine monomers Correction of pyrimidine dimers by DNA photolyase Base flipping

29. Fotoliase

30. Regulasi Repair: SOS Regulon

33. Mismatch repair- colon cancer • Penyebab hereditary nonpolyposis colon cancer (HNPCC) • Mutasi pada mikrosatelit –hMLH1, hMSH2 = MutS dan MutL pada E.coli • CC 4.8

34. Mismatch repair • Correct insertion or deletion up to 4 nt • Additional proofreading after Pol E. Coli DNA methylation site: A base in GATC sequence Newly synthesized DNA – hemimethylated MutS, MutL and MutH protein complex participate in the mismatch repair MutS recognize mismatch MutS2MutL2 recognize methylated GATC palindrome, and activates endonuclease MutH – nick DNA The unmethylated ssDNA is removed by UvrD protein The gap is filled by DNA polymerase Human DNA: Methylated in C position in CG sequence Homologs of MutS and MutL proteins (MSH and MLH) proteins Defect in mismatch repair system: Hereditary nonpolyposis colon cancer

35. Mismatch repair Beda untai lama dan baru ?

36. HUNTINGTON DISEASE CAG : GLUTAMIN NORMAL PENDERITA 36 -82 KOPI

37. HUNTINGTON DISEASE • CAG : GLUTAMIN • PENDERITA 36 -82 KOPI Fagile X syndrome: berpengaruh pada sistem regulasinya

39. O O O O O

42. O O

43. MUTAGEN

44. MUTAGEN KIMIAWI • BASA ANALOG • 5-Bromourasil dan 2-aminopurin bergabung pada DNA seperti asam nukleat lainnya • BU = analog timin, yang berpasangan dengan A tetapi juga dapat berpasangan dengan G • Terjadi mutasi dari A:T menjadi G:C

45. BROMURASIL DALAM BENTUK ENOL BU:G

46. AFLATOKSIN B1 • AFLATOKSIN B1 OLEH SITOKROM P450 DIUBAH MENJADI SENYAWA EPOKSID YG BEREAKSI DENGAN G • G:C MENJADI T:A

47. SENYAWA INI BEREAKSI DENGAN 7-N GUANOSIN menghasilkan radikal G:C MENJADI T:A

49. ASAM NITRIT • MENGOKSIDASI ADENIN (A) MENJADI HIPOSANTIN • HIPOSANTIN INI AKAN BERPASANGAN DENGAN C • A:T MENJADI G:C

50. A:T G:C