1. 1 Dr. Parvin Pasalar Tehran University of Medical Sciences دانشگاه علوم پزشكي وخدمات بهداشتي درماني تهران

2. 2 Objectives: To know and explain about:  Mutation and its causes  Different basis of Mutation classification  Mutagens  Positive aspects of mutations  Ames test  Repair systems  Diseases related to repair system defect

3. 3Mutation Definition: An un- repaired damages to DNA Causes: It may be spontaneous or induced because of different agents Classifications: are classified on different basis Their importance: Genetic Disease & raw material for the development

4. 4 Different Causes of mutations: Different Causes of mutations: Contrary to popular belief… Most DNA damage is caused by endogenous mutagens Estimated DNA damage/day in human cells SSBs ~50,000/day Depurinations ~10,000/day Deaminations ~600/day Oxidations ~2000/day Alkylations ~5000/day DSBs ~50-100/day

5. 5 What is the scale of our worries? We each have 46 chromosome = 6 X 10 9 bp DNA/cell On average, a mistake is made once every in 10 9 bp of DNA copied So, we have 6 mistakes/cell/division We have ~10 14 cells in our body that divide a minimum of once per year So, ~ 6 X 10 14 mistakes per year… Or, at least 60 billion mistakes while in this class today!!!

6. 6 Classifications of Mutations Spontaneous: At physiologic rate Induced: Because of the treatment with different agents 1- Can be spontaneous or induced 2- May be substitutions or frameshift 3- May occur in structural or regulatory sequences 4- May be small (point) or big 5- May have no or severe effect 6- Somatic or germinal Origins of Spontaneous Mutation Errors in DNA replication DNA polymerase accuracy Errors in DNA recombination DNA strands alignment Base alterations and base damage tautomerization; deamination; depurination; oxidation; alkylation Spontaneous frameshift mutations mispairing during replication and recombination Substitution: The base number remains the same but the types changes: -transitions: pu to pu or py to py -tranversion: pu to py or py to pu Frameshift: The base number changes -in coding regions, insertion or deletion of a nt that is not a multiple of 3 changes gene coding sequence Introduces premature stop codons=protein truncation Qualitative changes: Mutation structural sequences may causes changes in the sequence of aa of the resulting product (polypeptide) Quantative changes: Mutation in regulatory sequences does not change the structure of the product but its amount Small: Gene mutation are those that change a gene Small: Gene mutation are those that change a gene Big: Chromosomal rearrangements Big: Chromosomal rearrangements - can be inversions, deletions, - can be inversions, deletions, translocations, or amplifications translocations, or amplifications -can alter chromosome organization -can alter chromosome organization and affect gene function and affect gene function -can activate gene expression -can activate gene expression -can create novel fusion genes -can create novel fusion genes -can affect chromosome segregation -can affect chromosome segregation (non dysjunction) during meiosis-semi- sterility (non dysjunction) during meiosis-semi- sterility -some types of rearrangements in -some types of rearrangements in meiosis may be of evolutionary benefit meiosis may be of evolutionary benefit Classifications of substitution Mutations: Missense mutation Base pair substitution results in substitution of a different amino acid. Nonsense mutation Base pair substitution results in a stop codon (and shorter polypeptide). Neutral mutation Base pair substitution results in substitution of an amino acid with similar chemical properties (protein function is not altered). Silent mutation Base pair substitution results in the same amino acid (or nucleotide).

7. 7 Different Causes of Mutations Biological (normal error rate in DNA metabolic processes) Physical (Radiation) Sunlight Chemical (Mutagens, Carcinogens) 1- Alkylating agents 2- Base analogues 3- intercalating agents 4- Different chemicals such as: a- Nitrous acid b- Hydroxylamine

8. 8 1- Double-strand breaks (DSBs) 1- Double-strand breaks (DSBs) 2- Single- strand breaks (SSBs) 2- Single- strand breaks (SSBs) 3- Base alteration / damage 3- Base alteration / damage a: Oxidation a: Oxidation b: Alkylations b: Alkylations c: Hydrolysis c: Hydrolysis depurination depurination deaminations deaminations Different Type of DNA damages

9. 9 DNA Damage, Repair, and Consequences Damaging agentConsequences Repair Process In hibition of: Replication Transcription Chromosome segregation Mutation Chromosom e aberration

10. 10 a: Oxidation: It is caused by: 1- Normal metabolism 2- ROS (reactive oxygen species) such as O 2 -, H 2 O 2, OH. 3- Ionizing radiation 4- Chemicals It causes: Base-mispairing (i.e., oxoG can pair with C or A) Base alteration/damage

11. 11 Base alteration/damage b: Alkylation : It is caused by: Transfer of methyl or ethyl group to DNA bases It causes Base-mispairing (ie., O6-methylG mispairs with T)

12. 12 Types of base alterations and supressor tRNA

13. 13 Deamination Depurination C: Hydrolytic damage: Deamination: It is caused by : Conversion of amino groups of A, G, and C to keto groups. It causes: Changes in base pairing properties Depurination: It is caused by : Base loss (hydrolysis) It causes: -breaking of base: sugar bond -creates abasic site

14. 14 Induced Mutagenesis Physical (Radiation) UV Ionizing Chemical (Mutagens, Carcinogens) 1- Alkylating agents 2- Base analogues 3- intercalating agents 4- Different chemicals such as: a- Nitrous acid b- Hydroxylamine

15. 15 PHYSICAL MUTAGENS / RADIATION radiation was discovered in the 1890s -Roentgen discovered X-rays in 1895 -Becquerel discovered radiation in 1896 -Marie and Pere Curie discovered radioactive elements in 1898 first discovered mutagenic agent known -effects on genes first reported in 1920s in Drosophila (Muller) BIOLOGICALLY SIGNIFICANT EM spectrum -consists of electric and magnetic waves

16. 16 Sources of radiation: 1- Natural sources of radiation -cosmic, terrestrial, atmosphere 2- Anthropogenic -medical testing devices -nuclear testing and power plants -other products (TV’s, smoke detectors, Scanners) Types of radiation: Long wave length Visible UV Ionizing PHYSICAL MUTAGENS / RADIATION

17. 17 Definition: Wavelength < 320 nm Less energetic than IR (non-ionizing) It is preferentially absorbed: by aromatic compound It causes: covalent attachment of adjacent pyrimidines in one strand bulky lesions; can block replication, and transcription can stimulate mutation 1. Ultraviolet (UV) radiation Classification: UV-C: 180-290 nm, (germicidal) UV-B: 290-320 nm, (major lethal/mutagenic fraction in sunlight) UV-A: 320 nm-visible light (near UV; produces few pyrimidine dimers, but can produce reactive oxygen radicals)

18. 18 Definition: Wavelength < 180 nm More energetic than UV It produces ROS that : 1- react with DNA and other biological molecules. 2- Make breaks in one or both strands mutations and gross chromosomal rearrangements. 3- Increases recombination rate & death if unrepaired. 4- Crosslinking of DNA to itself or proteins. 5- ROS affects rapidly dividing cells & effects are dose- dependent. Classification: X rays Gamma rays 2. Ionizing radiation (IR)

19. 19 1- Base analogs: resemble purines and pyrimidines 1- Base analogs: resemble purines and pyrimidines bromouracil (BU) & aminopurine bromouracil (BU) & aminopurine CHEMICAL MUTAGENS

20. 20 CHEMICAL MUTAGENS They are: Flat, multiple ring molecules, that can interact with and insert between DNA bases. It Causes: DNA to be stretched Insertion of an extra base opposite intercalated molecule by DNA polymerase = FRAMESHIFT MUTATION acridine orange ethidium bromide proflavin 2- intercalating agents

21. 21 3- Nitrous acid: 3- Nitrous acid: cause deaminations cause deaminations C  U, meC  T C  U, meC  T A  hypoxanthine A  hypoxanthine 4-Nitrosoguanidine 4-Nitrosoguanidine cause base alkylation cause base alkylation methyl and ethyl methyl and ethyl methanesulfonate methanesulfonate 5-Hydroxylamine 5-Hydroxylamine Hydroxylates amino-gp of C Hydroxylates amino-gp of C C pairs with A C pairs with A CHEMICAL MUTAGENS

22. 22 1- Sterilization: Induction of mutation to sterile germs. 2- Making small changes in protein sequence. Site-specific in vitro mutagenesis is a method by which mutant alleles can be synthesized in the lab and transformed into cell culture and animals. Mutagenesis as a tool !

23. 23 Bruce Nathan Ames Brith:1928 Ames test: 1970 Can we detect Mutagen: Ames Assay

24. 24 Dr. Parvin Pasalar Tehran University of Medical Sciences دانشگاه علوم پزشكي وخدمات بهداشتي درماني تهران

25. 25

26. 26 Some questions: 1- How much of DNA synthesis in a prokaryote is because of Replication? 1- How much of DNA synthesis in a prokaryote is because of Replication? 2- Why DNA is double stranded? 2- Why DNA is double stranded? 3- Why we are diploid? 3- Why we are diploid?

27. 27 How to Repair & what is the un-repaired consequences Damaging agentConsequences Repair Process In hibition of: Replication Transcription Chromosome segregation Mutation Chromosom e aberration

28. 28 DNA Repair Pathways 4.Recombinational repair - multiple pathways - double strand breaks and interstrand cross-links 5.Tolerance mechanisms - lesion bypass - recombination 1. Direct reversals 2. Excision repair a. Base excision repair (BER) b. Nucleotide excision repair (NER) 3. Mismatch repair - replication errors

29. 29 Damage Recognized: Thymine dimers 6-4 photoproduct Gene Products Required: Photolyase Related disease: Photolyase not yet found in placental mammals TT Visible light TT 1- Direct reversal: photoreactivation

30. 30 a. Base Excision Repair damaged bases are removed as free bases primarily responsible for removal of oxidative and alkylation damages most genes in pathway are essential and have an important role in aging b. Nucleotide Excision Repair damaged bases are removed as oligonucleotides primarily responsible for removal of UV-induced damage and bulky adducts also removes ~ 20% of oxidative damage deficient in human disorders 2- Excision Repair Pathways

31. 31 DNAP+ Ligase DNA Ligase BER NER 2- Excision Repair Pathways

32. 32 3- Mismatch Repair( MMR) in E. coli : Decision between right & wrong (methyl-directed) Before replication both strands of GATC are methylated Shortly afte replication it is hemimethylated After a while it becomes fully methylated again

33. 33 3- MMR in E. coli Damage Recognized: Base-base mismatch (except C-C) Small insertion/deletion loops (IDLs ) Gene Products Required (11): MutS (damage recognition) MutL MutH (endonuclease) MutU (DNA helicase) Exonucleases (ExoI, ExoVII, ExoX, RecJ) DNA polymerase III Single strand binding protein (SSB) DNA Ligase

34. 34 Definition: Using of another DNA molecule( homologous) as template Function: The system is important in normal C.O When it is used : Double Strand Breaks & interstrand cross-links The consequence: Gene Conversion. 4- Recombinational repair Is defected in: Bloom ’ s Syndrome

35. 35 Summary

36. 36 Genetics of NER in Humans 1- Xeroderma Pigmentosum Occurrence: 1-4/10 6 population Sensitivity: sunlight Disorder: multiple skin disorders; malignancies of the skin neurological and ocular abnormalities Biochemical defect: early step of NER Genetic: seven genes (A-G), autosomal recessive

37. 37 2- Cockayne’s Syndrome Occurrence: 1 per/ 10 6 population Sensitivity: sunlight Disorder: arrested development, mental retardation, dwarfism, deafness, optic atrophy, intracranial calcifications Biochemical defect : NER Genetic: five genes (A, B and XPB, D & G) autosomal recessive Genetics of NER in Humans

38. 38 MMR Mutations in Hereditary Nonpolyposis Colon Cancer (HNPCC) MMR mutations in 70% of families MMR mutations in 70% of families Population prevalence 1: 2851 (15-74 years) Population prevalence 1: 2851 (15-74 years) 18% of colorectal cancers under 45 years 18% of colorectal cancers under 45 years 28% of colorectal cancers under 30 years 28% of colorectal cancers under 30 years