Gene Mutation, DNA Repair, and Transposition Lecture Notes PDF

GENE MUTATION, DNA REPAIR, AND TRANSPOSITION Maria Ysabel Morte, MD Michael Christian Virata, MD  CENTRAL DOGMA OF MOLECULAR BIOLOGY REVIEW GENE MUTATION ARE CLASSIFIED IN VARIOUS WAYS Mutation - Any base pair change in the part of DNA ⇢ alteration of nucleotide seque...

GENE MUTATION, DNA REPAIR, AND TRANSPOSITION Maria Ysabel Morte, MD Michael Christian Virata, MD  CENTRAL DOGMA OF MOLECULAR BIOLOGY REVIEW GENE MUTATION ARE CLASSIFIED IN VARIOUS WAYS Mutation - Any base pair change in the part of DNA ⇢ alteration of nucleotide sequence of an organism’s genome. ○ It can occur in somatic cells or within germ cells (basis of genetic diversity and evolution) CLASSIFICATION BASED ON MOLECULAR CHANGE Point mutation or Base substitution - A change of one base pair to another in a DNA molecule. Missense mutation ○ A change of one nucleotide of a triplet within a protein coding portion to a different amino acid resulting in the creation of a new triplet that codes for a different mutation OTHER TYPES OF POINT MUTATION Nonsense mutation ○ The triplet will be changed into a stop codon, resulting in the termination of the protein. Silent mutation ○ The point mutation alters a codon but does not result in a change in the amino acid at that position. OTHER TYPES OF POINT MUTATION Neutral mutation ○ Mutation that occurs in noncoding DNA ○ They do not affect gene products or gene expression. TRANSITION VS. TRANSVERSION Transition - If a pyrimidine replaces a pyrimidine or a purine replaces a purine Transversion - purine replaces pyrimidine, or vice-versa CLASSIFICATION BASED ON MOLECULAR CHANGE Frameshift mutation ○ Insertion or deletion of one or more nucleotides at any point within the gene. ○ Resulting in change of all the subsequent three-letter codons. ○ It is possible that one of the many altered triplets codons will be stop codon ⇢ halting polypeptide synthesis CLASSIFICATION BASED ON EFFECT ON FUNCTION Loss-of-function mutation - reduces or eliminates the function of the gene product Null mutation - complete loss of function Gain-of-function mutation - codes for a gene product with enhanced, negative, or new functions. Suppressor mutation - a second mutation that reverts or relieves the effects of a previous mutation. ○ Intragenic mutation - suppressor mutation occur within the same gene that suffered the first mutation ○ Intergenic mutation - different gene CLASSIFICATION BASED BY PHENOTYPE Example of conditional mutation. Mutation in black pigment enzyme works only on cooler temperature and stops at body temperature. Seen also in Himalayan rabbits Mutations occur spontaneously and randomly Spontaneous mutation ○ changes in the nucleotide sequence that occur naturally. ○ No specific agents are associated with their occurrence. ○ A result of normal biological or chemical process in the organism. ○ Example: DNA replication Mutations occur spontaneously and randomly MUTATION RATE - likelihood that a gene will undergo mutation in a single generation or in forming a single gamete 1. Rate of spontaneous mutation is exceedingly low for all organisms. 2. The rate varies between different organisms 3. Even within the same species, the spontaneous mutation rate varies from gene to gene. Spontaneous mutations arise from replication errors and base modification DNA replication is imperfect. DNA polymerase may insert incorrect nucleotides during replication of a strand of DNA. They possess 3’ to 5’ exonuclease. Tautomers - bases that increases the chance of mispairing during DNA replication. Replication slippage - Insertion or deletion that occur when one strand of the DNA template loops out (miss the nucleotides) and becomes displaced during replication, or when DNA polymerase slips or stutters during replication. SPONTANEOUS MUTATIONS EXAMPLES OF SPONTANEOUS MUTATIONS Depurination Loss of one of the nitrogenous bases in an intact double-helical DNA, most frequently guanine or adenine. Deamination An amino group in cytosine or adenine is converted to a keto group. Cytosine is converted to uracil and adenine is converted to hypoxanthine. Oxidative Reactive oxygen species (electrophilic oxidants) “free radicals”. It damage can produce different types of DNA chemical modification Transposable Jumping genes. Acts as a naturally occuring mutagen or can elements create chromosomal damage.  Mutations occur spontaneously and randomly Induced mutation ○ Results from influence of exogenous factors ○ Example: UV radiation causing skin cancer. Squamous cell carcinoma from chronic exposure in UV rays  INDUCED MUTATIONS EXAMPLES OF INDUCED MUTATIONS Mechanism Examples Base analogs Compounds that can substitute for purines or pyrimidines during nucleic 5-bromouracil acid biosynthesis. Alkylating They donate an alkyl group (CH3 or CH2CH3) to amino or keto groups in Mustard gases and agents nucleotides ⇢ altered base pairing affinities. ethylmethane sulfonate Intercalating Chemicals with different dimension and shapes allowing them to wedge Ethidium bromide agents between the base pairs of DNA ⇢ DNA replication, transcription and repair is altered ⇢ frame-shift mutations Adduct-forming A substance that covalently binds to DNA, altering conformation and Acetaldehyde agents interfering with replication and repair. (cigarette smoke)  INDUCED MUTATIONS EXAMPLES OF INDUCED MUTATIONS Mechanism Examples Ultraviolet Surface UV radiation can induce thousands of DNA lesions per UV rays from sun rays hour in any cell exposed to this radiation through creation of pyrimidine dimers distorting DNA conformation and inhibit normal replication Ionizing They transform stable molecules into free radicals Alpha, gamma radiation rays, and cosmic rays. SINGLE-GENE MUTATION & HUMAN DISEASES Most human genetic diseases are polygenic - OMIM - Online Mendelian Inheritance in caused by several genes but studies have Man database showed even a single base-pair change in ○ A comprehensive database of human one of the approximately 20,000 human genes, mutations, and disorders is genes can lead to a serious inherited disorder available online to be explored. Monogenic diseases - can be caused by ○ As of 2017, the OMIM database has many different types of single-gene catalogued approximately 6000 human mutations phenotypes for which the molecular basis is known SINGLE-GENE MUTATION & HUMAN DISEASES POINT MUTATION ○ Nonsense Mutations - 30% of the human mutations that can cause human disease are single base-pair changes Not only code for a prematurely terminated protein product, but also trigger rapid decay of mRNA ○ Missense Mutations - alter the amino acid sequence of a protein FRAMESHIFT MUTATION ○ alter the protein sequence and create internal nonsense codons.  SINGLE-GENE MUTATION & β-THALASSEMIA An inherited autosomal recessive blood disorder resulting from a reduction or absence of hemoglobin. Most common single-gene disease in the world, especially populations in: ○ Mediterranean, North African, Middle Eastern, Central Asian, Southeast Asian countries People with β-thalassemia have varying degrees of anemia ○ Severe to mild symptoms including: Weakness Delayed development Jaundice Enlarged organs Need for frequent blood transfusions Mutations in the β-globin gene (HBB gene) cause β- thalassemia ○ HBB gene encodes 146-amino acid β-globin polypeptide Types of mutations (missense, nonsense, and ○ Two β-globin polypeptides combined with two α-globin frameshift) that causes β-thalassemia not only polypeptides to form adult hemoglobin tetramer affect the β-globin amino acid sequence but also alter HBB transcription efficiency, mRNA splicing and stability, translation, and protein stability. EXPANDABLE DNA REPEATS Researchers observed some mutant genes contain an expansion of trinucleotide repeat sequences which as short DNA sequences repeated many times Normal individuals have a low number of repetitions of these sequences; however, individuals with over 20 different human disorders appear to have abnormally large numbers of repeat sequences When trinucleotide repeats such as (CAG) occur within a coding region, they can be translated into long tracks of glutamine causing proteins to aggregate abnormally Example of Diseases with Trinucleotide Repeat Expansions: Fragile-X Syndrome One of the most common causes of inherited intellectual disability affecting especially a person’s behavior and ability to learn Myotonic Dystrophy Characterized by progressive muscle wasting and weakness Huntington Disease Inherited disorder that causes nerve cells (neurons) to gradually break down and die DNA REPAIR SYSTEMS Living systems have evolved a variety of elaborate repair systems that counteract both spontaneous and induced DNA damage. These repair systems are absolutely essential to the maintenance of the genetic integrity of organisms and such to the survival of organisms on Earth. Tomas Lindahl, Paul Modrich, and Aziz Sancar has been awarded a Nobel Prize in Chemistry for their groundbreaking insights into the ways that cells detect and repair DNA damage. PROOFREADING AND MISMATCH REPAIR Some of the most common types of mutations arise during DNA replication when incorrect nucleotide is inserted by DNA polymerase DNA Polymerase III, the major DNA synthesizing enzyme in bacteria makes an error approximately once every 100,000 insertions. Fortunately, DNA polymerase proofreads each step, catching 99% of those errors ○ If an incorrect nucleotide is inserted… Recognizes the error ⇢ reverses its direction ⇢ behaves as 3’ to 5’ exonuclease ⇢ cuts incorrect nucleotide ⇢ replaces with correct nucleotide MISMATCH REPAIR (MMR) - another mechanism that may be activated to cope with errors such as base-base mismatches, small insertions, and deletions that remain even after proofreading POSTREPLICATION REPAIR Responds after damaged DNA has escaped repair and has failed to be completely replicated When DNA bearing a lesion of some sort (such as pyrimidine dimer) is being replicated, DNA polymerase may stall at the lesion and then skip over it, leaving an unreplicated gap on the newly synthesized strand. ○ RecA, a protein that directs recombinational exchange may correct the gap PHOTOREACTIVATION REPAIR: Reversal of UV Damage UV light introduces mutations by the creation of pyrimidine dimers UV-induced damage to E.coli DNA can be partially reversed if, following irradiation, cells are briefly exposed to visible light, especially blue range of the visible spectrum. ○ This process is dependent on the activity of photoreactivation enzyme (PRE) or photolyase which main mode of action is cleaving the cross-linking bonds between thymine dimers. BASE AND NUCLEOTIDE EXCISION REPAIR Another light-dependent DNA repair system in bacteria and eukaryotes Basic mechanism involved in these types of repair is collectively referred to as excision repair or cut-and-paste mechanism ○ STEPS: 1. Presence of damage, distortion, or error on one of two strands of the DNA helix is recognized and enzymatically clipped out by an endonucleases. Excision usually includes number of nucleotides adjacent to the error as well, leaving a gap on one strand. 2. DNA Polymerase fills in the gap by inserting nucleotides complementary to those on the intact strand. 3. DNA ligase seals the nick. BASE EXCISION REPAIR (BER) - corrects DNA that contains incorrect base pairings due to the presence of chemically modified bases or uridine nucleosides that are inappropriately incorporated into DNA or created by deamination of cytosine. NUCLEOTIDE EXCISION REPAIR (NER) - pathway repair “bulky” lesions in DNA that alter or distort the double helix. NER mechanism in eukaryotes is much more complicated than that in bacteria involving many more proteins, encoded by about 30 genes. XERODERMA PIGMENTOSA (XP) A recessive genetic disorder caused by defects in NER pathways and other mutations in more than one gene that predisposes individuals to severe skin abnormalities, skin cancers, and a wide range of other symptoms including developmental and neurological defects. Patients with XP are also extremely sensitive to UV radiation in sunlight. In addition, they have a 2000-fold higher rate of cancer (skin cancer). The condition is severe and maybe lethal, although early detection and protection from sunlight can arrest it.  AMES TEST Named after the American biochemist Bruce Ames who invented the assay in the 1960s A test that is used to assess mutagenicity of compounds such as synthetic chemicals found: ○ Air and water pollution ○ Food preservatives ○ Artificial sweeteners ○ Herbicides ○ Pesticides ○ Pharmaceutical products Initial Ames Testing in the 1970s examined a large number of known carcinogens showing 80% of which are strong mutagens - 60 compounds in cigarette smoke tests positive TRANSPOSABLE ELEMENTS LEADING TO MUTATIONS Transposable Elements (TEs), informally known as “jumping genes” are DNA sequences that can move or transpose within and between chromosomes, inserting themselves into various locations within the genome. ○ Can range from 50 to 10,000 base pairs in length ○ Has been known to be a significant source of evolution but can also be a great source of mutations DNA TRANSPOSONS - moves from one location to another without going through an RNA intermediate stage which are very abundant in many organisms ○ Requires: Inverted terminal repeats (ITRs) on each end of the TE and an Open reading frame (ORF) codes for the enzyme transposase; for movement TRANSPOSABLE ELEMENTS LEADING TO MUTATIONS Transposable Elements (TEs), informally known as “jumping genes” are DNA sequences that can move or transpose within and between chromosomes, inserting themselves into various locations within the genome. ○ Can range from 50 to 10,000 base pairs in length ○ Has been known to be a significant source of evolution but can also be a great source of mutations RETROTRANSPOSONS - TEs that amplify and move within the genome using RNA as an intermediate. Method of transposition are sometimes described as “copy-and-paste” mechanism. ○ Retrotransposons resemble retroviruses in many ways, such in which replication uses a similar mechanism but Retrotransposons do not encode all of the proteins required to form mature virus particles and therefore are not infective. End of lecture Next: Movie Paper Analysis  William, S. Klug, W.S, Cummings, M.R., Spence,r C., Palladino, M.A., Kil- lian, D. (2021). Concepts of REFERENCE/S: Genetics (12th ed.)