Mutations Lecture Slides PDF

Mutations DNA Damage & Repair Mechanisms MGD Module Jan 10, 2024 Shara K. Zheer Nizamaddin [email protected] 1  Learning Objectives 1. Explain relationship between changes in nucleotide...

Mutations DNA Damage & Repair Mechanisms MGD Module Jan 10, 2024 Shara K. Zheer Nizamaddin [email protected] 1  Learning Objectives 1. Explain relationship between changes in nucleotide & amino acid sequences 2. Describe different types of mutational changes, e.g. point mutation, indel 3. Predict & explain effect that different mutations may have 4. Describe how spontaneous & induced mutations may occur 5. Describe genetic link between mutation & mutant & explain how some mutations can be inherited 6. Describe process & role of DNA repair 7. Explain relationship between DNA damage & Cancer 8. Recognize fundamental importance of PCR in the diagnosis of genetic disease. 2  What is mutation? Is any permanent, heritable change in DNA base sequence of an organism Mutations that result from damage to the nucleotides of DNA molecules or from unrepaired errors during replication can be transcribed into mRNA and therefore can, sometimes, result in the translation of a protein with an abnormal amino acid sequence It may cause genetic diseases or cell death They can also cause changes in enzyme activity, nutritional requirements, antibiotic susceptibility, and many other properties of cells So source of genetic variation, evolution, beneficial mutation, NOT always bad 3  Mutations: are a source of genetic variation üA mutation causes a mutant phenotype, which is a phenotype that differs from common or wild type phenotype in the population üA mutation in a gene causes a mutant allele, which is an allele that differs from common allele in the population (the wild type allele) üIt could be in one allele heterozygous or both allele homozygous 4 Mutation can be categorized to: 1. Small mutations: Change in nucleotide sequence of Single gene 2. Large scale mutations: (Will be covered in next lecture) a) Abnormal number of chromosome or b) Chromosome structure that are strictly abnormal 5  Types of small mutation- single Gene Mutations 1. Substitutions (Point mutations): A single base (nucleotide) change. -If in the coding region of a gene, it would be one of these: A. Silent: a mutation that does not alter the amino acid specified CGA (Arg) >>> CGG (Arg) B. Missense: a mutation that replaces one amino acid with another CGA (Arg) >>> CCA (Pro) C. Nonsense: a mutation that changes the amino acid specified to a stop codon CGA (Arg) >>> UGA (Stop) Base substitutions and can be either: ü Transition (purine to purine OR pyrimidine to pyrimidine). OR ü Transversion (purine to pyrimidine OR pyrimidine to purine). 6 7 - If in non-coding regions of the gene Point mutations in non-coding regions or outside genes can of course also be detrimental as they can change protein binding sites, promoter sequences, splice sites etc. 8 2. Insertion, Deletion, & frameshift mutations: An insertion or deletion of one or more bases to the DNA sequence If in the coding region of a gene A- Non- frameshift Insertion or deletion of 3 nucleotides (or multiples of 3): no change in reading frame (however, there will be a change in the amino acid code) +3 (GTT) ATG GAA GCA CGT ATG GAA GTT GCA CGT Met Glu Ala Gly Met Glu Val Ala Gly B- Frameshift Insertion or deletion of nucleotides other than 3 or multiples of 3 (1, 2, 4, 5….) The reading frame shifts at the point where the insertion or deletion begins. Beyond that point, the amino acid sequence of the protein translated from mRNA Differs from the normal protein. -1 (G) ATG GAA GCA CGT ATG AAG CAC GT… Met Glu Ala Gly Met Lys His 9 Q1/A type of mutation that affect every codon beyond the point of mutation. a. Point Mutation b. Silent Mutation c. Nonsense Mutation d. Frameshift Mutation Q2/ A non-conservative mutation is one in which: a. The new amino acid has different biochemical properties than the original b. A frameshift has occurred, causing production of a new amino acid c. A large segment of DNA is deleted d. No effect results in the protein Q3/ Which type of mutation results in the sickle-cell disease phenotype? (Mutation results in glutamate →→ valine) a. Conservative mutation b. Frameshift mutation c. Non-conservative missense mutation d. Codon deletion 10 Predict and explain the EFFECT of mutation (Disease) Its based on type mutation and location in the gene Is it in the coding region or in the regulatory region? If in the coding region: Point mutation? what kind of point mutation? del/Inser? Is it three or a multiple of three? If not so its frameshift If in the regulatory region/non coding region Point mutation-Which region of the gene? del/Inser- Which region of the gene? Also is it small or large del/insertion 11 Type of mutation- in coding region Effect on Protein Silent: new codon specifies same None amino acid Missense: new codon specifies Possible decrease in function; variable -Its either conservative or different amino acid effects. depends on chemical properties of nonconservative. the new amino acid Sickle cell – nonconservative Factor XI deficiency Nonsense: new codon is stop codon Shorter than normal; usually nonfunctional Non frameshift insertion/deletion Possible decrease in function; variable Cystic fibrosis – Deletion of CTT at effects. depends on function of the amino exon 11 acid added or deleted Frameshift insertion/deletion Usually nonfunctional; often shorter than Gaucher disease, an inserted normal single DNA base prevents production of an enzyme Type of mutation- in regulatory Effect on Protein region/non-coding region Point mutations Does not affect quality of protein, but may Becker muscular dystrophy- affect quantity-Affect rate of transcription protein dystrophin is normal, but its levels are reduced Insertion or deletion Does not affect quality of protein, but may Some mutations of β+ 12 affect quantity. Affect rate of transcription thalassemia Example on effects of mutation Q/ Saman has been having recurrent infections and build-up of thick, sticky mucus in his lung. His physician suspected Cystic fibrosis (CF), therefore, a sample was taken/sent for PCR genetic analysis to check for CTT deletion in CFTR gene (the most common type of mutations). CFTR gene encoded for a transmembrane protein that maintaining the balance of salt and water on many cell surfaces in the body, including lung. Healthy: 5‘-...ATC ATC TTT GGT GTT TCC TAT GAT GAA TAT....-3’ CF: 5‘-...ATC ATT GGT GTT TCC TAT GAT GAA TAT....-3’ CFTR protein: N-...ile ile phe gly val ser tyr asp glu tyr...-C Answer the following? a) Although more than 1000 mutations are identified in CFTR gene, why the doctor asked for CTT deletion? b) Is the CTT deletion a frameshift or non-frameshift mutation? c) Why the loss of CTT (Phenylalanine) in the protein sequence of that clinical significance? d) Was it the right choice to use PCR technique for the detection of CTT deletion? 13 e) Since CF is an autosomal recessive disorder, accordingly to the PCR- gel electrophoresis, which of the samples is Saman’s sample and why? 1 2 3 14 Q/ Azad was tested for β thalassemia. His DNA sample was sequenced for β globin gene. The gel shown below is for the region that normal & mutant sample (Azad’s sample is different. The mutation is homozygous, in the non coding region- promoter. What type of mutation is it? How does it affect the severity of the disease GGA TCC CGA GGT CCC GAT GG TCC CGA 15 Causes of Mutations 16 1- Spontaneous Mutation 2. Induced Mutation: non-replication dependent damage DNA Replication error Tautomeric shift- mismatch (TG or CA) DNA Polymerase slippage- Trinucleotide repeat expansion mutation -Results in many neurological conditions (Huntington's disease- ploy- glutamine repeat expansion instead of 6-35 it changes to 36-121) 17  2- Induced mutation In addition to spontaneous mutations, problems may arise from DNA damaged by mutagens: chemicals produced in cells, inhaled, or absorbed from environment that cause mutations, either directly or indirectly. Exposure to Mutagens can be: Intentional: used in Medicine for treatment e.g radiation (Gamma rays) and chemotherapy (base analog, interlacing agent) to kill cancer cells Unintentional: UV light, X-Ray, tabaco X-Ray: generating free radical OH. upon hitting water in cells, reacts with DNA and damage it. UV light: causes pyrimidine dimers between two adjacent Thymine on DNA. Mutations may result in melanomas, appearing as dark brown growths on the skin Smoking mutagen- Tabaco (cigarettes and shisha) when it burns, form a bulky compound bind to G, prevent GC base pairing 18 Mutation can affect chromosomes in: 1- Somatic cells- called somatic mutations Are passed on to successive generations of cells, but only within an organism (i.e. horizontal transmission) 2- Gametes-called germline mutations Inherited & transmitted to offspring, vertical transmission of hereditary disease *It is becoming apparent that a number of diseases—& perhaps most cancers—are due to the combined effects of vertical as well as horizontal transmission of induced mutations 19 How our cells deal with all these errors & mutagens exposure on a daily basis? DNA Repair Mechanisms 20 DNA Damage, Repair, & Cancer 21  Identifying & Repair Mechanism Mutation is unavoidable Both normal metabolic activities in human cell and environmental factors can cause DNA damage Its crucial to identify and repair the damages The cell has several highly conserved mechanisms in place to check and repair the genetic code: During DNA replication After completion of replication Mutations happen very frequently but are being recognized and repaired very frequently too Failure of DNA repair can have serious consequences to the cell, and can cause disease. 22 During DNA Replication Proofreading by DNA Polymerase Additional activities occur during replication include proofreading & DNA repair Pol δ also has the 3` to 5` exonuclease activity required for proofreading. Enzymes that catalyze repair of mismatched bases are also present Consequently, eukaryotic replication occurs with high fidelity; approximately one mispairing occurs for every 109 to 1012 nucleotides incorporated into growing DNA chains 23 Repair Mechanisms Both normal metabolic activities in human cell and environmental factors can cause DNA damage, therefore, there is a collection of processes by which a cell identifies and corrects damage to the DNA molecule. These are: 1. EXCISION REPAIR a) NUCLEOTIDE b) BASE 2. MISMATCH REPAIR The three repair mechanisms have steps in common, shown in the next slide. 24 25 1-Excision Repair -In base excision repair, the glycosylase enzyme cleaves the glycosidic bond between altered base and ribose. -In nucleotide excision repair, the entire nucleotide is removed at once by excision endonuclease enzyme. The repair involves damages from chemical carcinogens, cigarettes, UVB in sunlight (thymine dimer) or oxidative damage from free radical by X ray. 26  Xeroderma Pigmentosum (XP), An autosomal recessive. Cause by defect/deficiency of endonuclease enzyme (mutation in at least 9 genes) in nucleotide excision repair mechanism. This allow thymine dimers formed upon exposure to sun UV light stay & block replication. In normal individual, usually repair mechanisms correct this damage, and cancer rarely occurs. But in XP cancer extremely common, because of the inability to repair DNA, the frequency of mutation increases. A cancer develops once proto-oncogenes or tumor suppressor genes mutate. By avoiding light, these individuals can reduce the number of skin cancers that develop. 27  2-Mismatch Repair (MMR) DNA mismatch repair is a system for recognizing and repairing incorrect insertion, deletion, and mis- incorporation of bases that can arise during DNA replication and recombination, as well as repairing Mismatched bases (e.g A-C instead of A-T or G_C) are recognized by enzymes of the mismatch repair system, MLH1 and other protiens. 28  Hereditary nonpolyposis colorectal cancer (HNPCC) Also called Lynch syndrome An autosomal dominant inheritance Caused by mutations in one of genes of DNA mismatch repair system, mainly MLH1 gene. MLH1 mutation confers a markedly increased risk for various types of cancer, particularly of the colon and the endometrium. 29  DNA Damage, Repair, & Cancer References Genetics A Conceptual Approach; Benjamin A. Pierce. Fourth edition, 2012 Kaplan Medical Genetics USMLE step 1 Chapter 4 Edition 2021 30 Lippincott’s Illustrated Reviews: Biochemistry 5th edition Chapter 5