Lecture 14 Mutation & Repair II Fall 2024 BIOL 23373 PDF

BIOL 23373 – General Genetics Fall 2024 Lecture 14 Mutation & Repair II Announcements If you want to meet (in person or via Zoom), email me 3 or 4 meeting day/time options. Tutoring Tutoring @ the CORD offers 1-on-1 and small group assistance in over 10...

BIOL 23373 – General Genetics Fall 2024 Lecture 14 Mutation & Repair II Announcements If you want to meet (in person or via Zoom), email me 3 or 4 meeting day/time options. Tutoring Tutoring @ the CORD offers 1-on-1 and small group assistance in over 100 U of A courses. Students can meet with tutors in person or online by scheduling an appointment or accessing drop-in services. Learn more about Tutoring services and book an appointment at: https://success.uark.edu/academic-initia tives/tutoring.php Corresponding Readings Chapter sections: 15.1-15.7 Gene Mutations May Arise Spontaneously Spontaneous mutations arise in cells without exposure to agents capable of inducing mutation These arise primarily through errors in DNA replication or spontaneous changes in the chemical structure of a nucleotide base Spontaneous mutations provide a background mutation rate that varies among species and among genes 5 DNA Replication Errors DNA replication has very high fidelity due to proofreading ability of DNA polymerases Genomic regions containing repeat sequences experience higher levels of error during replication These result in changes in number of base pairs in repeating sequences (i.e., the number of repeats) 6 DNA Repeat Mutations Alterations in number of DNA repeats occur via strand slippage DNA polymerase temporarily dissociates from the template and a portion of the newly replicated DNA forms a temporary hairpin Resumption of replication leads to re-replication of some of the repeats and an overall increase in the number of repeats on the daughter strand 7 DNA Repeat Mutations Spontaneous Nucleotide Base Changes DNA nucleotides can occasionally convert to alternative structures called tautomers with slight differences in bonding and placement of hydrogens 9 Spontaneous Nucleotide Base Changes Tautomeric shifts can lead to base-pair mismatches and incorporation of incorrect bases during replication This is the most common form of replication error 10 Mutations May Be Induced by Chemicals or Radiation Induced mutations are produced by interactions between DNA and physical, chemical, or biological agents that generate mutations Elevate mutation rate above background rate Agents that cause DNA damage leading to mutations are called mutagens Mutagens interact with DNA in specific ways to cause particular types of sequence changes Chemical mutagens can be classified by how they modify DNA 11 Chemical Mutagens Chemical mutagens can be classified by their mode of action on DNA as: 1. nucleotide base analogs-similar structure to DNA nucleotide 2. deaminating agents-remove amino groups from nucleotide 3. alkylating agents-add side groups such as methyl (CH3) and ethyl (CH3-CH2) to nucleotides 4. oxidizing agents-add oxygen or remove hydrogen atom from nucleotide 5. hydoxylating agents-add hydroxyl group to nucleotide 6. intercalating agents-insert between DNA base pairs 12 Chemical Mutagens 13 The Ames Test The Ames Test mimics what happens when animals are exposed to chemicals, and tests chemicals and their breakdown products for mutagenic potential Bacteria (usually Salmonella typhimurium) are exposed to experimental compounds in the presence of mammalian liver enzymes In animals, ingested chemicals are transported to the liver, where they are broken down by enzymes 14 The Ames Test Procedure Bacterial strains carry mutations interfering with their ability to synthesize histidine (his) Bacteria are exposed to the chemical to be tested, plus an extract of purified liver enzymes, and plated on a medium lacking histidine A chemical is mutagenic if there is The number of revertants a significant increase in the reversion rate in treated strains from his to his are relative to controls assayed for each treatment or control 15 Electromagnetic Spectrum Ionizing Non-ionizing Gamma (γ) rays X-rays U IR Microwaves Radio waves V 10-16 10-14 10-12 10-10 10-8 10-6 10-4 10-2 100 102 104 106 108 Wavelength, λ (m) 400 700 nm nm 16 UV light Pyrimidine dimers are produced by the formation of one or two additional covalent bonds between adjacent pyrimidine nucleotides 17 Ionizing Radiation X-rays, gamma rays, cosmic rays Short wavelength, high energy; can penetrate deeply into tissues Causes widespread damage and double-stranded breaks, which can lead to chromosomal aberrations Increases risk of leukemia and other cancers 18 Repair Systems Correct Some DNA Damage The integrity of DNA is under continuous assault from spontaneous changes and from changes induced by mutagens Organisms preserve the fidelity of DNA using multiple repair systems that either directly repair DNA damage or allow the organism to circumvent the problems caused by unrepaired damage 19 Repair of DNA Damage The most direct way to repair DNA damage is to identify and then reverse the changes One mechanism is the proofreading activity of DNA polymerase (see Lecture 6 – DNA Replication II) Additional repair systems include: Mismatch repair Photoreactivation repair Base excision repair Nucleotide excision repair Post-replication repair 20 Mismatch Repair Errors after proofreading by the DNA polymerase can be fixed by the mismatch repair mechanism. Mismatch repair involves several steps: 1. Endonuclease nicks the new strand with error 2. Exonuclease removes bases on nicked strand 3. DNA polymerase synthesis fills the gap 4. DNA ligase seals the gap 21 Photoreactivation Repair Pyrimidine dimers due to UV exposure can be repaired via photoreactivation repair, in bacteria, single-celled eukaryotes, plants, and some animals (but not humans) The enzyme photolyase uses energy from visible (blue) light to break the bonds between pyrimidine dimers 22 Base Excision Repair Base excision repair corrects mispairings due to chemical modification of bases DNA glycosylases recognize and remove mispaired nucleotides by cleaving the modified base from the sugar, leaving an apurinic or apyrimidinic (AP) site AP endonuclease then removes the remainder of the nucleotide DNA polymerase and DNA ligase fill and seal the gap with the appropriate nucleotide 23 Nucleotide Excision Repair If UV-induced damage cannot be directly repaired, it can be excised and replaced Two UVR-A protein and one UVR-B bind the DNA strand opposite the damage, then UVR-A proteins dissociate One UVR-C joins UVR-B and cleaves phosphodiester bonds on the damaged DNA strand on both sides of damage– about 12-13 nucleotides apart UVR-D (a helicase) unwinds DNA to release damaged segment DNA polymerase synthesizes the missing nucleotides and DNA ligase seals the gap 24 Post-replication Repair Large-scale DNA damage can overwhelm the ability of repair systems DNA replication can bypass damaged sections, leaving single-stranded gaps Recombination repair directs recombination of the incomplete segment with the complementary strand, using the sister chromatid as the repair template 25 Missense Mutation: Sickle-cell Disease 26 An Inherited Hemoglobin Variant Causes Sickle Cell Disease Sickle-cell disease (SCD) is a potentially fatal, autosomal recessive disorder caused by a structural abnormality in hemoglobin (Hb) that affects its ability to carry oxygen SCD patients experience severe muscle pain when sickle-shaped blood cells in circulation are numerous enough to impede blood flow in smaller vessels; reduced blood flow deprives surrounding tissues of oxygen leading to pain and tissue damage Sickled RBCs are removed from the circulatory system, but often cannot be replaced quickly enough, leading to chronic anemia 27 Hemoglobin Structure Hemoglobin molecules are tetramers with two protein chains each of two different globin genes: a-globin and b-globin This arrangement is the most common form and is called hemoglobin A (HbA) Each of the four proteins carry one iron-containing molecule of heme, which reversibly binds to oxygen 28 The Globin Genes The a-globin and b-globin genes are organized similarly with three exons and two introns The a-globin gene encodes a protein 141 amino acids long and b-globin encodes one 146 amino acids in length 29 Sickle-cell Disease SCD is a common hereditary anemia caused by a single base-pair substitution in the b-globin gene that changes an amino acid The mutant allele is called b S and the normal allele is called b A Individuals with SCD carry two b S alleles and have genotype b S/b S 30 Cause of Sickle Shape in Red Blood Cells When two abnormal b-globin proteins join two normal a-globin proteins, the hemoglobin molecules are structurally and functionally abnormal Instability of the abnormal hemoglobin molecules can cause them to collapse into linear crystal-like molecules, causing the deformation of the red blood cells into the characteristic sickle shape 31 Heterozygous Individuals Carry the Mutant Allele Heterozygous individuals (carriers of SCD allele) have genotype b A/b S Some of their hemoglobin molecules carry defective b-globin proteins Because most of their red blood cells are normal, they do not develop severe anemia They are sometimes identified as having the sickle 32 Sickle-cell Disease in Human Populations Dozens of variant alleles of hemoglobin genes produce one form or another of hereditary anemia Most of these are rare, but a few are found in high frequency in some populations The  S allele is found in frequencies up to 15% in several populations of Africa, the Middle East, India, and Madagascar. The high frequencies of mutant alleles suggest that natural selection maintains their prevalence 33 Malaria As an Agent of Natural Selection An environment where malaria is endemic favors the survival and reproduction of individuals who are heterozygous for  A and one of the mutant alleles Malaria, a potentially fatal disease, is caused by protozoans, commonly Plasmodium falciparum, carried by the mosquito, Anopheles gambeii, which transfers the parasite to animals it bites Symptoms of malaria include high fever and can cause death if untreated; once infected, a person can suffer recurrences throughout life 34 Malaria As an Agent of Natural Selection Individuals who are homozygous succumb more easily to malaria ( A/ A) or suffer from hereditary anemia ( S/ S) Heterozygotes with genotype  A/ S survive and reproduce better than other genotypes in regions where malaria is common The advantage heterozygotes derive is based on the shorter average life span of their RBCs that thwarts the malaria parasite via interrupting the developmental cycle of Plasmodium 35 Heterozygote Advantage and Equilibrium Frequency Heterozygote advantage causes populations to evolve a gene pool with high frequencies of both the normal and mutant alleles The advantage of heterozygosity is balanced by the disadvantages associated with homozygosity for either allele (i.e., balancing/stabilizing selection) The conflicting pressures lead to equilibrium frequencies of normal and mutant alleles in population 37 Evidence for Heterozygote Advantage 1. The frequency of SCD carriers rises with increasing age in a population, due to earlier death of homozygotes 2. Heterozygous women produce larger numbers of children, on average 3. Across the “malaria belt” the  S allele has arisen and evolved independently at least three times 38 X