Lecture 5 BBT317 Mutation and Repair Part V.pptx

BBT317 Molecular Genetics Lecture # 5  Mutation and Repair: Part V Pierce Genetics: A Conceptual Approach (4e or 5e) Chapter 18 Snustad Principles of Genetics (6e) Chapter 13 Klug Essentials of Genetics (10e) Chapter 14 Watson Molecular Biology of the Gene (7e) Chapter 10 Griffiths...

BBT317 Molecular Genetics Lecture # 5  Mutation and Repair: Part V Pierce Genetics: A Conceptual Approach (4e or 5e) Chapter 18 Snustad Principles of Genetics (6e) Chapter 13 Klug Essentials of Genetics (10e) Chapter 14 Watson Molecular Biology of the Gene (7e) Chapter 10 Griffiths Introduction to Genetic Analysis (11e) Chapter 16  Conditional Lethal Mutations Conditional Lehtal Mutations: Powerful Tools for Genetic Studies (Snustad Chapter 13) Conditional lethal mutation is any mutation that produces a mutant (conditional lethal mutant) whose viability depends on the conditions of growth. It grows normally in permissive conditions but in restrictive conditions it does not grow, thereby expressing its lethal mutation. The three major classes of mutants with conditional lethal phenotypes are (1) auxotrophic mutants, (2) temperature-sensitive mutants, and (3) suppressor-sensitive mutants. Q1. What are conditional lethal mutations? Q2. What are the three major classes of mutants with conditional lethal phenotypes?  Conditional Lethal Mutations Three Major Classes of Conditional Lethal Mutants (Snustad Chapter 13) Auxotrophs are mutants that are unable to synthesize an essential metabolite (amino acid, purine, pyrimidine, vitamin, and so forth) that is synthesized by wild-type or prototrophic organisms of the same species. The auxotrophs will grow and reproduce when the metabolite (for example, histidine) is supplied in the medium (the permissive condition); they will not grow when the essential metabolite (for example, histidine) is absent (the restrictive condition). Temperature-sensitive mutants will grow at one temperature but not at another. Most temperature-sensitive mutants are heat-sensitive; however, some are cold- sensitive. The temperature sensitivity usually results from the increased heat or cold lability of the mutant gene product—for example, an enzyme that is active at low temperature but partially or totally inactive at higher temperatures. Suppressor-sensitive mutants are viable when a second genetic factor, a suppressor, is present, but they are nonviable in the absence of the suppressor. The suppressor gene may correct or compensate for the defect in phenotype that is caused by the suppressor-sensitive mutation, or it may cause the gene product altered by the mutation to be nonessential. Q1. Discuss the three major classes of conditional lethal mutants?  Conditional Lethal Mutations Three Major Classes of Conditional Lethal Mutants (Literature) Temperature-sensitive mutants Suppressor-sensitive mutants The three termination codons, UAG, UAA, and UGA are also known as amber, ochre, and opal. http://cospi.iiserpune.ac.in/TSpred/Utility.html https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9008745/#:~:text=Mechanism%20of%20amber%20suppression.,from%20a%20GUC%20to%20AUC. No question in exam on this slide.  Screening for mutagens Evaluating Mutagens in our Environment by Ames Test Q1. What is the importance of the Ames test?  Screening for mutagens Evaluating Mutagens in our Environment by Ames Test (Klug) The Ames test (named for American biochemist Bruce Ames of the University of California Berkeley, who invented the assay in the 1960s) uses a number of different strains of the bacterium Salmonella typhimurium that have been selected for their ability to reveal the presence of specific types of mutations. For example, some strains are used to detect base-pair substitutions, and other strains detect various frameshift mutations. Each strain contains a mutation in one of the genes of the histidine operon. The mutant strains are unable to synthesize histidine (his- strains) and therefore require histidine for growth. The assay measures the frequency of reverse mutations that occur within the mutant gene, yielding wild-type bacteria (his+ revertants) (Figure 14.14). The his- strains also have an increased sensitivity to mutagens due to the presence of mutations in genes involved in both DNA damage repair and the synthesis of the lipopolysaccharide barrier that coats these bacteria and protects them from external substances. Many substances entering the human body are relatively innocuous until activated metabolically, usually in the liver, to more chemically reactive products. Thus, the Ames test includes a step in which the test compound is incubated in vitro in the presence of a mammalian liver extract. Alternatively, test compounds may be injected into a mouse where they are modified by liver enzymes and then recovered for use in the Ames test. Q1. Explain the Ames test to detect  Screening for mutagens Evaluating Mutagens in our Environment by Ames Test (Klug) In the initial use of Ames testing in the 1970s, a large number of known carcinogens, or cancer- causing agents, were examined, and more than 80 percent of these were shown to be strong mutagens. This is not surprising, as the transformation of cells to the malignant state occurs as a result of mutations. For example, more than 60 compounds found in cigarette smoke test positive in the Ames test and cause cancer in animal tests. Although a positive response in the Ames test does not prove that a compound is carcinogenic, the Ames test is useful as a preliminary screening device, as it is a rapid, convenient way to assess mutagenicity. The Ames test is used extensively during the development of industrial and pharmaceutical chemical compounds. Q1. Explain the Ames test to detect  Screening for mutagens Evaluating Mutagens in our Environment by Ames Test (Literature) https://www.youtube.com/watch?app=desktop&v=DkBye6HGtOc No question in exam on this slide.  DNA Repair Mechanisms DNA Repair Pathways The most important DNA repair mechanism is the proofreading function of the DNA polymerases that replicate DNA as part of the replisome. Both DNA polymerase I and DNA polymerase III are able to excise mismatched bases that have been inserted erroneously. Other DNA repair pathways are: Q1. What are the DNA repair pathways? DNA Repair Mechanisms DNA Repair Pathways (Pierce) Q1. What are the common DNA repair mechanisms?  DNA Repair Mechanisms Direct Reversal of Damaged DNA (Griffiths, Snustad) The most straightforward way to repair a lesion is to reverse it directly, thereby regenerating the normal base. Although most types of damage are essentially irreversible, lesions can be repaired by direct reversal in a few cases. Direct DNA Repair Pathways: 1. Photo-reactivation or Light-dependent repair (in bacteria, for example, E. coli) 2. Direct repair by Alkyltransferases Photo-reactivation or Light-dependent repair (in bacteria, for example, E. coli) Light-dependent repair or photoreactivation of DNA in bacteria is carried out by a lightactivated enzyme called DNA photolyase. When DNA is exposed to ultraviolet light, thymine dimers are produced by covalent cross-linkages between adjacent thymine residues. DNA photolyase recognizes and binds to thymine dimers in DNA, and uses light energy to cleave the covalent cross-links (Figure 13.25). Photolyase will bind to thymine dimers in DNA in the dark, but it cannot catalyze cleavage of the bonds joining the thymine moieties without energy derived from visible light, specifically light within the blue region of the spectrum (>300 nm). Q1. What are the direct DNA repair pathways?  DNA Repair Mechanisms Direct Reversal of Damaged DNA (Snustad) Q1. Discuss the photoreactivation pathway for DNA repair.  DNA Repair Mechanisms Direct Reversal of Damaged DNA (Pierce, Griffiths) Direct repair by Alkyltransferases Direct repair also corrects O6-methylguanine, an alkylation product of guanine that pairs with adenine, producing G · C→T · A transversions. An enzyme called O6-methylguanine-DNA methyltransferase removes the methyl group from O6- methylguanine, restoring the base to guanine (Figure 18.26). This enzyme transfers the methyl group from O6-methylguanine to a cysteine residue in the enzyme’s active site. However, the transfer inactivates the enzyme, and so this repair system can be saturated if the level of alkylation is high enough. Q1. Discuss the role of alkyltransferases in direct DNA repair.  DNA Repair Mechanisms Excision Repair Pathways: Base-excision repair (Pierce) In base-excision repair, a modified base is first excised and then the entire nucleotide is replaced. The excision of modified bases is catalyzed by a set of enzymes called DNA glycosylases, each of which recognizes and removes a specific type of modified base by cleaving the bond that links that base to the 1′-carbon atom of deoxyribose sugar (Figure 18.27a). Uracil glycosylase, for example, recognizes and removes uracil produced by the deamination of cytosine. Other glycosylases recognize hypoxanthine, 3-methyladenine, 7-methylguanine, and other modified bases. After the base has been removed, an enzyme called AP (apurinic or apyrimidinic) endonuclease cuts the phosphodiester bond, and other enzymes remove the deoxyribose sugar (Figure 18.27b). DNA polymerase then adds one or more new nucleotides to the exposed 3′-OH group (Figure 18.27c), replacing a section of nucleotides on the damaged strand. The nick in the phosphodiester backbone is sealed by DNA ligase (Figure 18.27d), and the original intact sequence is restored (Figure 18.27e). Q1. Discuss the base excision repair pathway for damaged DNA.  DNA Repair Mechanisms Excision Repair Pathways: Base-excision repair (Pierce) Q1. Discuss the base excision repair pathway for damaged DNA.  DNA Repair Mechanisms Excision Repair Pathways: Base-excision repair (Pierce) Bacteria use DNA polymerase I to replace excised nucleotides, but eukaryotes use DNA polymerase β, which has no proofreading ability and tends to make mistakes. On average, DNA polymerase β makes one mistake per 4000 nucleotides inserted. About 20,000 to 40,000 base modifications per day are repaired by base excision, and so DNA polymerase β may introduce as many as 10 mutations per day into the human genome. How are these errors corrected? Recent research results show that some AP endonucleases have the ability to proofread. When DNA polymerase β inserts a nucleotide with the wrong base into the DNA, DNA ligase cannot seal the nick in the sugar–phosphate backbone, because the 3′-OH and 5′-P groups of adjacent nucleotides are not in the correct orientation for ligase to connect them. In this case, AP endonuclease 1 detects the mispairing and uses its 3′→5′ exonuclease activity to excise the incorrectly paired base. DNA polymerase β then uses its polymerase activity to fill in the missing nucleotide. In this way, the fidelity of base-excision repair is maintained. Q1. Which enzyme does the replacement of excised nucleotides during base excision repair of damaged DNA? What are its limitations?  DNA Repair Mechanisms Excision Repair Pathways: Nucleotide-excision repair (Griffiths, Pierce, Snustad) Although the vast majority of the damage sustained by an organism is minor base damage that can be handled by base-excision repair, this mechanism can neither correct bulky adducts that distort the DNA helix, adducts such as the cyclobutane pyrimidine dimers caused by UV light, nor correct damage to more than one base. A DNA polymerase cannot continue DNA synthesis past such lesions, and so the result is a replication block. A blocked replication fork can cause cell death. Similarly, an abnormal or damaged base can stall the transcription complex. To cope with both of these situations, prokaryotes and eukaryotes utilize an extremely versatile pathway called nucleotide-excision repair (NER) that is able to relieve replication and transcription blocks and repair the damage. It is found in cells of all organisms from bacteria to humans and is among the most important of all repair mechanisms. In NER, a unique excision nuclease activity produces cuts on either side of the damaged nucleotide(s) and excises an oligonucleotide containing the damaged base(s). This nuclease is called an excinuclease to distinguish it from the endonucleases and exonucleases that play other roles in DNA metabolism. Q1. What is the importance of the nucleotide excision repair pathway? Q2. What is an excinuclease?  DNA Repair Mechanisms Excision Repair Pathways: Nucleotide-excision repair (Snustad) The E. coli nucleotide excision repair pathway is shown in Figure 13.27. In E. coli, excinuclease activity requires the products of three genes, uvrA, uvrB, and uvrC (designated uvr for UV repair). A trimeric protein containing two UvrA polypeptides and one UvrB polypeptide recognizes the defect in DNA, binds to it, and uses energy from ATP to bend the DNA at the damaged site. The UvrA dimer is then released, and the UvrC protein binds to the UvrB/DNA complex. The UvrC protein cleaves the fourth or fifth phosphodiester bond from the damaged nucleotide(s) on the 3’ side and the eighth phosphodiester linkage from the damage on the 5’ side. The uvrD gene product, DNA helicase II, releases the excised dodecamer. In the last two steps of the pathway, DNA polymerase I fills in the gap, and DNA ligase seals the remaining nick in the DNA molecule. Q1. Discuss the nucleotide excision repair pathway of E. coli.  DNA Repair Mechanisms Excision Repair Pathways: Nucleotide-excision repair (Snustad) Q1. Discuss the nucleotide excision repair pathway of E. coli.  DNA Repair Mechanisms Excision Repair Pathways: Nucleotide-excision repair (Snustad) Nucleotide excision repair in humans occurs through a pathway similar to the one in E. coli, but it involves about four times as many proteins. In humans, the excinuclease activity contains 15 polypeptides. Protein XPA (for xeroderma pigmentosum protein A) recognizes and binds to the damaged nucleotide(s) in DNA. It then recruits the other proteins required for excinuclease activity. In humans, the excised oligomer is 24 to 32 nucleotides long rather than the 12-mer removed in E. coli. The gap is filled in by either DNA polymerase δ or ε in humans, and DNA ligase completes the job. Q1. What is the difference with respect to the nucleotide excision repair pathway between bacteria and humans?  DNA Repair Mechanisms Post-replication repair: Mismatch Repair Pathways (Griffiths) Q1. Discuss the mismatch repair pathway of E. coli.  DNA Repair Mechanisms Post-replication repair: Mismatch Repair Pathways Q1. Discuss the mismatch repair pathway of E. coli.  DNA Repair Mechanisms Post-replication repair: Mismatch Repair Pathways (Snustad) MutH contains a GATC-specific endonuclease activity that cleaves the unmethylated strand at hemimethylated GATC sites either 5’ or 3’ to the mismatch. The incision sites may be 1000 nucleotide pairs or more from the mismatch. If the incision occurs at a GATC sequence 5 to the mismatch, a 5 → 3 exonuclease like E. coli exonuclease VII is required. If the incision occurs 3 to the mismatch, a 3 → 5 exonuclease activity like that of E. coli exonuclease I is needed. After the excision process has removed the mismatched nucleotide from the unmethylated strand, DNA polymerase III fills in the large—up to 1000 bp—gap, and DNA ligase seals the nick. Q1. Which protein in the E. coli mismatch repair pathway has the GATC-specific endonuclease activity? Q2. What are the functions of exonuclease VII and exonuclease I in E. coli mismatch repair pathway? Q3. Discuss the conservation of mismatch repair pathway.  DNA Repair Mechanisms Post-replication repair: Mismatch Repair Pathways (Watson) Q1. Discuss the directionality in mismatch repair.  DNA Repair Mechanisms Post-replication repair: Mismatch Repair Pathways (Watson) Homologues of the E. coli MutS and MutL proteins have been identified in fungi, plants, and mammals—an indication that similar mismatch repair pathways occur in eukaryotes. In fact, mismatch excision has been demonstrated in vitro with nuclear extracts prepared from human cells. Thus, mismatch repair is probably a universal or nearly universal mechanism for safeguarding the integrity of genetic information stored in double-stranded DNA. Q1. Discuss the conservation of mismatch repair pathway.  Next Lecture: Mutation and Repair Part VI