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Mutation

Mutation. دانشگاه علوم پزشكي وخدمات بهداشتي درماني تهران. Dr. Parvin Pasalar Tehran University of Medical Sciences. Mutation. Definition: An un- repaired damages to DNA Causes: It may be spontaneous or induced because of different agents Classifications:

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Mutation

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

  2. Mutation • Definition: Anun- repaired damagesto DNA • Causes: It may be spontaneousor induced because of different agents • Classifications: are classified on different basis • Their importance: Genetic Disease& raw material for the development

  3. Different Causes of mutations: Contrary to popular belief… Most DNA damage is caused by endogenousmutagens 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 -

  4. What is the scale of our worries? • We each have 46 chromosome = 6 X 109 • bp DNA/cell • On average, a mistake is made once • every in 109 bp of DNA copied • So, we have 6 mistakes/cell/division • We have ~1014 cells in our body that • divide a minimum of once per year • So, ~ 6 X 1014 mistakes per year… Or, at least 60 billion mistakes while in class for Biochemistry today!!!

  5. Classifications of Mutations 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 Types of mutations in ORFs: 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). Frameshift mutations: Deletions or insertions (not divisible by 3) result in translation of incorrect amino acids, stops codons (shorter polypeptides),or read-through of stop codons (longer polypeptides). 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 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 Spontaneous: At physiologic rate Induced: Because of the treatment with different agents • 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 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 • Big: Chromosomal rearrangements - can be inversions, deletions, translocations, or amplifications -can alter chromosome organization and affect gene function -can activate gene expression -can create novel fusion genes -can affect chromosome segregation (non dysjunction) during meiosis-semi- sterility -some types of rearrangements in meiosis may be of evolutionary benefit

  6. 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

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

  8. DNA Damage, Repair, and Consequences Damaging agent Consequences • In hibition of: • Replication • Transcription • Chromosome segregation • Mutation • Chromosome aberration Repair Process

  9. Base alteration/damage a: Oxidation: It is caused by: 1- Normal metabolism 2- ROS (reactive oxygen species) such as O2-, H2O2, OH. 3- Ionizing radiation 4- Chemicals It causes: Base-mispairing (i.e., oxoG can pair with C or A)

  10. Basealteration/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)

  11. Base alteration/damage Deamination Types of base alterations • 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 Depurination Deamination

  12. 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

  13. PHYSICAL MUTAGENS / RADIATION EM spectrum -consists of electric and magnetic waves • 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

  14. PHYSICAL MUTAGENS / RADIATION • 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

  15. 1. Ultraviolet (UV) radiation • 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 • 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)

  16. 2. Ionizing radiation (IR) • 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

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

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

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

  20. Mutagenesis as a tool ! 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.

  21. Can we detectMutagen: Ames Assay Bruce Nathan Ames Brith:1928 Ames test: 1970

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

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

  24. How to Repair & the un-paired consequences Damaging agent Consequences • In hibition of: • Replication • Transcription • Chromosome segregation • Mutation • Chromosome aberration Repair Process

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

  26. 1-Direct reversal: photoreactivation T T T T Visible light Damage Recognized: Thymine dimers 6-4 photoproduct Gene Products Required: Photolyase Related disease: Photolyase not yet found in placental mammals

  27. 2-Excision Repair Pathways • 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 • thought to 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

  28. 2-Excision Repair Pathways BER NER DNA Ligase DNAP+ Ligase DNAP+ Ligase

  29. Genetics of NER in Humans 1- Xeroderma PigmentosumOccurrence: 1-4/106 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

  30. Genetics of NER in Humans 2- Cockayne’s Syndrome Occurrence: 1 per/ 106 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

  31. 3- Mismatch Repair 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

  32. 3- Mismatch Repair (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

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

  34. 4- Recombinational repair • 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. • Defect in Human:Bloom’s Syndrome

  35. Summary

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