Chapter 7: DNA Mutation, DNA Repair and Transposable Elements
Chapter 7: DNA Mutation, DNA Repair and Transposable Elements -
Chromosomal mutations – changes involving whole chromosomes or sections of them
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Point mutation – a change of one or a few base pairs – may change the phenotype of the organism if it occurs within the coding region of a gene or in the sequences regulating the gene
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Gene mutations – affect gene function - can alter a phenotype by changing the function of the protein
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Transposable elements – genetic change occurring when certain mobile genetic elements in chromosome move from one location to another in the genome
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Transposition – change in position
DNA Mutation Adaptation versus Mutation -
Adaptation – the environment induced an adaptive inheritable change
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Some geneticists thought that variation among organisms resulted from random mutations that sometimes happened to be adaptive
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Others thought that variations resulted from adaptation; that is, the environment induced an adaptive inheritable change
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The Adaptation Theory – Lamarckism – the doctrine of the inheritance of acquired characteristics
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Keynote: heritable adaptive traits result from random mutation, rather than by adaptation as a result of induction by environmental influences
Mutations Defined -
Mutation – is the process by which the sequence of base pairs in a DNA molecule is altered – may result in a change to either a DNA base pair or a chromosome
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Somatic mutation – (in multicellular organisms) the mutant characteristic affects only the individual in which the mutation occurs and it not passed on to the succeeding generation
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Germ-line mutation – may be transmitted by the gametes to the next generation, producing an individual with the mutation in both its somatic and its germ-line cells
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Mutation rate – probability of a particular kind of mutation as a function of time, such as the number of mutations per nucleotide pair per generation
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Mutation frequency – the number of occurrences of a particular kind of mutation, expressed as the proportion of cells or individuals in a population; such as the number of mutations per 100,000 organisms
Types of Point Mutations -
Base-pair substitutions – a change from one base pair to another in DNA. Depending on how the gene is translated to amino acid sequences it can result in no change to the protein, an insignificant change or a noticeable change. Two types:
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(1) a transition mutation – from one purine-pyrimidine base pair to the other purinepyrimidine base pair, such as A-T to G-C; this means that the purine on one strand of the DNA is changed to the other purine, while the pyrimidine ion the complementary strand is changed to the other pyrimidine
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(2) a transversion mutation – a mutation from a purine-pyrimidine base pair to a pyrimidine-purine base pair, such as G-C to C-G or A-T to C-G
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Missense mutation – is a gene mutation in which a base-pair change causes a change in an mRNA codon so that a different amino acid is inserted into the polypeptide – a phenotypic change may or may not result, depending on the amino acid change involved
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Nonsense mutation – is a gene mutation in which a base-pair change alters an mRNA codon for an amino acid to a stop (nonsense) codon (UAG, UGA, UAA) – this causes premature termination of polypeptide synthesis, so shorter than normal polypeptide fragments (often non-functional) are released from the ribosomes
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Neutral mutation – is a base pair change in a gene that changes a codon in the mRNA such that the resulting amino acid substitution produces no detectable change in the function of the protein translated from that message – a neutral mutation is a subset of missense mutations in which the new codon codes for a different amino acid that is chemically equivalent to the original or the amino acid is not functionally important and therefore does not affect the proteins function
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Silent mutation - also known as a synonymous mutation – is a mutation that changes a base pair in a gene, but the altered codon in the mRNA specifies the same amino acid in the protein – in this case the protein obviously has wild type function – silent mutations most often occur by changes such as this at the third-wobble-position of a codon
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Frameshift mutation – usually results in a non-functional protein – if one or more base pairs are added or deleted from a protein-coding gene, the reading frame of mRNA can change downstream of the mutation – therefore incorrect amino acids are added to the polypeptide chain after the mutation – these mutations may generate new stop codons, resulting in a shortened or lengthened polypeptide chains
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Keynote: mutation is the process by which the sequence of base pairs in a DNA molecule is altered. Mutations that affect a single base pair of DNA are called base-pair substitutions mutations. Base-pair substitutions and single base-pair insertions or deletions are called point shift mutations. Mutations in the sequences of genes are called gene mutations
Reverse Mutations and Suppressor Mutations -
Point Mutations are divided into two classes based on how they affect phenoty[es:
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(1) a forward mutation – changes a wild type phenotype gene to a mutant gene
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(2) a reverse mutation – also known as a reversion or back mutation – changes a mutant gene at the same site so that it functions in a completely wild-type or nearly wild type way
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Reversion of a nonsense mutation occurs when a base pair change results in a change of mRNA nonsense codon to a codon for an amino acid. if this reversion is back to the wild type amino acid, the mutation is a true reversion – if the reversion is to some other amino acid, the mutation is a partial reversion, and complete or partial may be restored, dpending on the change
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Suppressor mutation – a mutation at a different site from that the original mutation. A suppressor mutation masks or compensates for the effects of the initial mutation, but does not reverse the original mutation
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Intragenic (in the same gene but different site) and intergenic (in a different gene from the original) suppressors – operate to decrease or eliminate deleterious effects of the original mutation.
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Intragenic suppressors – act by altering a different nucleotide in the same codon where the original mutation occurred or by altering a nucleotide in a different codon
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Intergenic suppression is the result of a second mutation in another gene. Genes that cause the suppression of mutations are called suppressor genes.
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Keynote: reverse mutations occur at the same site as the original mutation and cause the genotype to change from mutant to wild type. A suppressor mutation is one that occurs at a second site and completely or partially restores a function that was lost or altered because of a primary mutation. Intragenic suppressors are suppressor mutations that occur within the same gene where the original mutation occurred, but at a different site. Intergenic suppressors are suppressor mutations that occur in a suppressor gene – a gene different from the one with the original mutation.
Spontaneous and Induced Mutations -
Mutagenesis – the creation of mutations, can occur spontaneously or can be induced.
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Spontaneous mutations – are naturally occurring mutations
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Induced mutations – occur when an organism is exposed to either deliberately or accidental to a physical or chemical agent, known as a mutagen, that interacts with DNA to cause a mutation
Spontaneous Mutations -
All types of point mutations occur spontaneously
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They can occur during DNA replication, as well as during other stages of cell growth or division
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They can also result from the movement of transposable genetic elements
DNA Replication Errors -
Tautomers – when a base chemically can exist in alternative states – when a base changes its state it has undergone tautomeric shift – in DNA the keto form of each base is responsible for the normal Watson-Crick base pairing of T with A and C with G – however non-Watson-Crick base pairing can result in a base is in a rare tautomeric state, the enol form.
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Small additions and deletions can occur spontaneously during replication, they occur because of displacement-looping out- of bases from either the template or the growing DNA strand, generally in regions where a run of the same base or of a repetitive sequence is present. If DNA loops out from the template strand, DNA polymerase skips the looped-out base or bases, producing a deletion mutation; if DNA polymerase synthesizes an untemplated base or bases, the new DNA loops out from the template, producing an addition. An addition or deletion mutation un the coding region of a structural gene is a frameshift mutation if it involves other that 3 bp or a multiple of 3 bp
Spontaneous Chemical Changes -
Depurination – the loss of a purine from the DNA when the bond hydrolyzes between the base and the deoxyribose sugar, resulting in an apurinic site. – it occurs because the covalent bond between the sugar and the purine is much less stable than the bond between the sugar and the pyrimidine and is prone to breakage
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If such lesions are not repaired, there is no base to specify a complementary base during DNA replication, and the DNA polymerase may stall or dissociate from the DNA
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Deamination – is the removal of an amino group from a base – for example the deamination of cytosine produces uracil
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A repair system replaces most of the uracils in DNA thereby minimizing mutational consequences of cytosine deamination
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However if uracil is not replaces an adenine will be incorporated into the new DNA strand opposite it during replication, eventually resulting in a CG to TA transition mutation.
Induced Mutations -
Mutations can be induced by exposing organisms to physical mutagens, such as radiation, or to chemical mutagens. Deliberately induced mutations have played an important role in the study of mutations. Since the rate of spontaneous mutation is so low, mutagens are used to increase the frequency of a mutation so that a significant number of organisms have mutations in the gene being studied.
Radiation -
Radiation occurs in non-ionizing or ionizing forms. Ionization occurs when energy is sufficient to knock an electron out of an atomic shell and hence break covalent bonds. Except for UV, non-ionizing radiation does not induce mutations; but all forms of ionizing radiation such as X-rays, cosmic rays and radon can induce mutations
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UV light causes mutations by increasing the chemical energy of certain molecules, such as pyrmidines, in DNA. One effect of UV radiation on DNA is the formation of abnormal chemical bonds between adjacent pyrimidine molecules in the same strand of the double helix. This bonding is induced mostly between adjacent thymines, forming what are called thymine dimers (TT, CC, CT, TC). These unusual pairings produce a bulge in the DNA strand and disrupts the normal pairing of T with A. Replication cannot proceed past the lesion so the cell will die if enough pyrimidine dimmers remain unrepaired.
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Further (clarify) – ionizing radiation penetrates tissues, colliding with molecules and knocking electrons out of orbits, thereby creating ions. The ions can result in the breakage of covalent bonds, including those in the sugar-phosphate backbone of DNA.
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High dosages of ionizing radiation kills cells – hence their use in treating some forms of cancer. At certain low levels of ionizing radiation, point mutations are commonly produced; at these levels, there is a linear relationship between the rate of point mutations and the radiation dosage
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X-rays, Radon and Uranium are all sources of radiation and can all induce mutations
Chemical Mutagens -
Chemical mutagens include both naturally occurring chemicals and synthetic substances
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3 groups based on their mechanisms of action: base analogs, base-modifying analogs and intercalating agents
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Base analogs and intercalating agents depend on replication
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Base modifying agents can induce mutations at any point of the cell cycle
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Base analogs – are bases that are similar to those normally found in DNA. Like normal bases, base analogs exist in normal and rare tautomeric states. In each of the two states, the base analog pairs with a different base in DNA. Because base analogs are so similar to the normal nitrogen bases, they may be incorporated into DNA in place of the normal bases.
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An example of a base analog: 5-bromouracil (5 BU), which has a bromine residue instead of the methyl group of thymine. In its normal state 5 BU resembles thymine and pairs with adenine in DNA. In its rare state, it pairs with guanine. 5 BU induces mutations by switching between its two chemical states once the base analog has been incorporated into the DNA. If 5 BU is incorporated in its normal state, it pairs with adenine. If it then changes into its rare state during replication, it pairs with guanine instead. In the next round of replication, the 5 BU-G base pair is resolved into a C-G base pair instead of the T-A base pair. By this process a transition mutation is produced. 5 BU can also induce a CG-to-TA transition mutation if it is first incorporated into DNA in its rare state and then switches to the normal state during replication. Thus, 5 BUinduced mutations can be reverted by a second treatment of 5 BU.
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Base-modifying agents – are chemicals that act as mutagens by modifying the chemical structure and properties of bases. There’s three types of mutagens that work in this way: a deaminating agent, a hydroxylating agent, and an alkylating agent o
Nitrous acid, HNO2 is a deaminating agent that removes amino groups (-NH2) from the bases G, C and A. Treatment of guanine with nitrous acid produces xanthine, but because this purine base has the same pairing properties as guanine, no mutation results. Treatment of cytosine with nitrous acid produces uracil, which pairs with adenine to produce a CG-to-TA transition mutation during replication. Likewise, nitrous acid modifies adenine to produce hypoxanthine, a base that pairs with cytosine rather than thymine, which results in an AT-GC transition mutation. Therefore, a nitrous acid-induced mutation can be reverted by a second treatment with nitrous acid.
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Hydroxylamine (NH2OH) Is a hydroxylating mutagen that reacts specifically with cytosine, modifying it by adding a hydroxyl group (OH) so that it pairs with adenine instead of guanine. Mutations induced by hydroxylamine can only be CG-to-TA transitions, so hydroxylamine-induced mutations cannot be reverted by a second treatment with this chemical =. However, they can be reverted by treatment with other mutagens (such as 5 BU and nitrous acid) that cause TA-toCG transition mutations.
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Methylmethane sulfonate (MMS) is one of a diverse group of alkylating agents that introduce alkyl groups (-CH3, -CH2, CH3) onto the bases at a number of locations. Most mutations caused by alkylating agents result from the addition of an alkyl group to the 6-oxygen of guanine to produce O6-methylguanine. They methylated guanine pairs with thymine rather than cytosine, giving GC-to-AT transitions.
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Intercalating agents – such as proflavin, acridine and ethidium bromide (commonly used to stain DNA in gel electrophoresis experiments)- insert (intercalate) themselves between adjacent bases in one or both strands of the DNA double helix causing the helix to relax. If the intercalating agent inserts itself between adjacent base pairs of the DNA strand that is the template for new DNA synthesis, an extra base (chosen at random) is inserted into the new DNA strand opposite the intercalating agent. After one more round of replication, during which the intercalating agent is lost, the overall result is a base pair addition mutation. If the intercalating agent inserts itself into the new DNA strand in place of a base, then when that DNA double helix replicates after the intercalating agent is lost, the result is a base-pair deletion mutation. – if a base-pair addition or deletion point mutation occurs in a protein-coding gene, the result is a Frameshift mutation.
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KEYNOTE: mutations can be produced by exposure to chemical mutagens. If the genetic damage caused by the mutagen is not repaired, mutations result. Chemical mutagens act in a variety of ways, such as by substituting for normal bases during DNA replication, modifying the bases chemically, and intercalating themselves between adjacent bases during replication.
Site-specific in Vitro Mutagenesis of DNA -
Site-specific mutagenesis – mutating a gene at specific positions in the base-pair sequence in the test tube and then introduce the mutated gene back into the cell and investigate the phenotypic changes produced by the mutation in vivo.
Environmental Mutagens -
Our exposure to chemicals occurs primarily through eating food, absorption through the skin, and inhalation.
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For a mutagenic chemical to cause DNA changes it must enter cells and penetrate the nucleus, which many chemicals cannot do.
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Some chemicals are converted from mutagenic to non-mutagenic by our metabolism.
The Ames Test: A Screen for Potential Mutagens -
Carcinogens – chemical agents that induce mutations that result in tumorous or cancerous growth
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The mutations are typically base-pair substitutions that produce missense or nonsense mutations, or base pair additions or deletions that produce Frameshift mutations
The Ames test – assays the ability of chemicals to revert mutant strains of the bacterium Salmonella typhimurium to the wild type -
The Ames test, approximately 108 cells of tester bacteria that are auxotrophic for histidine (his mutants) are spread with or without mixture of rat, mouse, or hamster liver enzymes on a culture plate lacking histidine. Histidine (his) auxotrophs require histidine
in the growth medium in order to grow; normal (his+) individuals do not. An array of tester bacterial strains are available that allow detection of base-pair substitution mutations and frameshift mutations in the test. The liver enzymes called the S9 extract, are used because, many chemicals are not mutagenic themselves but are metabolized to mutagens (and carcinogens) in the body, often in the liver and other tissues. -
Test chemical is placed on the plate which is incubated over night and examined for colony formation the following day – a positive result in the Ames test is a significantly higher number of revertants near the test chemical disk than is seen on the control plate.
Detecting Mutations -
Mutations of haploid organisms are readily detectable because there is only one copy of the genome.
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Dominant mutations are also readily detectable, and X-linked recessive mutations can be detected because they are expressed in half of the sons of a mutated, heterozygous female. However, autosomal recessive mutations can be detected only if the mutation is homozygous.
Visible Mutants -
Visible mutants – affect the morphology or physical appearance of an organism. Examples of mutants are eye color, wing shape mutants, coat-color (like in albinos), colony size mutants of yeast, and plaque morphology mutants of bacteriophages.
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Since visible mutants are readily apparent, screening is done by inspection
Nutritional Mutants -
An auxotrophic (nutritional) mutant is unable to make a particular molecule essential for growth
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Auxotrophic mutants are most readily detected in microorganisms such as E. coli and yeast that grow on simple defined growth media from which they synthesize the molecules essential to their growth. A number of selection and screening procedures are available to isolate auxotrophic mutants.
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Replica plating – can be used to screen for auxotrophic mutants of any microorganism that grows in discrete colonies on a solid medium – samples from a culture of mutagenized or an unmutangenized colony-forming organism or cell type are plated onto a medium containing the nutrients appropriate for the mutants desired.
Conditional Mutants -
Conditional mutants – reduce the activity of gene products only under certain conditions – a common type of conditional mutation is a temperature-sensitive mutant (in yeast) normally grow at 23, r not at all at 36 degrees in order to be isolated. – heat
sensitivity typically results from a missense mutation causing a change in the amino acid sequence of a protein so that, at the higher temperature, the protein assumes a nonfunctional shape Resistance Mutants -
Mutations can be induced for resistance to particular viruses, chemicals, or drugs.
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For example: in E. coli mutants resistant to phage T1 have been induced and some mutants are resistant to antibiotics
KEYNOYE: A number of screening procedures have been developed to isolate mutants of interest from a heterogeneous mixture of cells in a mutagenized population of cells. Repair of DNA Damage -
Mutagenesis involves damage to DNA
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Especially with high dosages of mutagens, the mutational damage can be considerable. What we see as mutations are DNA alterations that are not corrected by various DNA damage repair systems – (mutations = DNA damage – DNA repair)
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There are two general categories of repair systems based on the way they function: direct reversal repair systems correct damaged areas by reversing the damage, whereas excision repair systems cut out a damaged area and then repair the gap by new DNA synthesis.
Direct Reversal of DNA Damage -
Mismatch Repair by DNA Polymerase Proofreading
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When an incorrect nucleotide is inserted, the polymerase often detects the mismatched base pair and corrects the area by backspacing to remove the wrong nucleotide and the resuming synthesis in the forward direction.
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The mutator mutations in E. coli illustrate the importance of the 3’ to 5’ exonuclease activity of DNA polymerase fir maintaining a low mutation rate. Mutator mutants have a much higher than normal mutation frequency for all genes. These mutants have mutations in genes for proteins whose normal functions are required for accurate DNA replication.
Repair of UV-induced Pyrimi=dine Dimers -
Photoreactivation or light repair – UV light induced thymine (or other pyrimidine) dimmers are reverted directly to the original form by exposure to near UV-light in the wavelength range from 320 to 370 nm. Photoreactivation occurs when an enzyme called photolyase is activated by a photon of light and splits the dimmers apart. Strains with mutations in the phr gene are defective in light repair. Phtolyase has been found in bacteria and in simple eukaryotes but not in humans,
Repair of Alkylation Damage -
Alkylating agents transfer alkyl groups (usually methl or ethyl groups) onto the bases. The mutagen MMS methylates the oxygen of carbon-6 in guanine. The enzyme removes the methyl group from the guanine, thereby chaning the base back to its original form. A similar specific system exists to repair alkylated thymine. Mutations of the genes encoding these repair enzymes result in a much higher rate of spontaneous mutations.
Excision Repair of DNA Damage -
Many mutations affect only one of the two strands. In such cases, the DNA damage can be excised and the normal strand used as a template for producing a corrected strand. Depending on the damage, excision may involve a single base or nucleotide, or two or more nucleotides. Each excision repair system involves a mechanism to recognize the specific DNA damage it repairs.
Base Excision Repair -
Damaged single bases or nucleotides are most commonly repaired by removing the base ot the nucleotide involved and the inserting the correct base or nucleotide
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Base excision repair – a repair glycosylase enzyme removes the damaged base from the DNA by cleaving the bond between the base and the deoxyribose sugar. Other enzymes then cleave the sugar-phosphate backbone before and after the now baseless sugar, releasing the sugar and leaving the gap in the DNA chain. The gap is filled with the correct nucleotide by a repair DNA polymerase and DNA ligase, with the opposite DNA strand used as the template. Mutations caused by depurination or deamination are examples of damage that may be repaired by base excision repair.
Nucleotide Excision Repair -
Dark repair or excision repair system (nucleotide excision repair – NER) – because the normal photoreactive repair system cannot operate n the dark, investigators hypothesized that there must be another light-independent repair system.
Methyl-Directed Mismatch Repair -
Methyl-Directed Mismatch Repair – correcting mismatched base pairs left after DNA replication – the system recognizes the incorrect bases, and then carries out repair synthesis
Translesion DNA Synthesis and the SOS Response -
Lesions that block the replication machinery from proceeding past that point can be lethal if unrepaired
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Translesion DNA synthesis – allows replication to continue past the lesions. The process involves a special class of DNA polymerases that are synthesized only in response to
DNA damage. In E. coli such DNA damage activates a complex system called SOS response (called SOS because it is a last resort, emergency response to mutational damage). The SOS response allows the cell to survive otherwise lethal events, although often at the expense of generating new mutations. KEYNOTE: Mutations constitute damage to the DNA. Both prokaryotes and eukaryotes have a number of repair systems that deal with different kinds of DNA damage. All the systems use enzymes to make the correction. Without such repair systems, lesions would accumulate and be lethal to the cell or organism. Not all lesions are repaired, and mutations do appear, but at low frequencies. At high doses of mutagens, repair systems are unable to correct all of the damage, and cell death may result. Human Genetic Diseases Resulting from DNA Replication and Repair Mutations -
Some human genetic diseases are attributed to defects in DNA replication or repair
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Example: xeroderma pigmentosum (OMIM 278700) is caused by homozygosity for a recessive mutation in a repair gene. Individuals with this lethal affliction are photosensitive, and portions of their skin that have been exposed to light show intense pigmentation, freckling and warty growths that can become malignant.
Transposable Elements General Features of Transposable Elements -
Transposable elements are normal, ubiquitous components of the genomes of prokaryotes and eukaryotes. Transposable elements fall into two general classes based on how they move from location to location in the genome. One class- found in both prokaryotes and eukaryotes – moves as a DNA segment. Members of the other class – found only in eukaryotes – are related to retroviruses and move via an RNA. First an RNA copy is synthesized; then a DNA copy of that RNA is made, and it integrates at a new site in the genome.
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In bacteria, transposable elements can move to new positions on the same chromosome (because there is only one chromosome) or onto plasmids or phage chromosomes
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In Eukaryotes, transposable elements may move to new positions within the same chromosome or to a different chromosome.
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In both bacteria and eukaryotes, transposable elements insert new chromosome locations with which they have no sequence homology; therefore, transposition is a process different from homologous recombination (recombination between matching DNA sequences) and is called nonhomologous recombination.
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Transposable elements are important due to the genetic changes they cause – for example, they can produce mutations by inserting into gene regulatory sequences (such as disrupting promotes on the element), and they can produce various kinds of chromosomal mutations through the mechanics of transposition.
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The frequency of transposition, though typically low, varies with the particular element, if the frequency were high, the genetic changes caused by the transpositions would likely kill the cell
Transposable Elements in Bacteria -
Two examples of transposable elements in bacteria are insertion sequence(IS) elements and transposons (Tn)
Insertion Sequences -
Insertion Sequences or IS element, is the simplest transposable element found in bacteria. An IS element contains only genes required to mobilize the element and insert it into a new location in the genome. IS elements are normal constituents of bacterial chromosomes and plasmids.
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IS elements were first identified in E. coli as a result of their effects on the expression of three genes that control the metabolism of the sugar galactose. Some mutations affecting the expression of these genes did not have properties typical of point mutations or deletions, but rather had an insertion of approx 800 bps DNA segment into a gene. This particular DNA segment is now called insertion sequence 1, or IS1, and the insertion of IS1 into the genome is an example of a transposition event.
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When IS elements integrate at random points along the chromosome, they often cause mutations by disrupting either the coding sequence of a gene or a gene’s regulatory region. Promoters within IS elements themselves may also have effects by altering the expression of nearby genes.
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In addition, the presence of an IS element in the chromosome can cause mutations such as deletions and inversions in the adjacent DNA
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Finally deletion and insertion events can also occur as a result of crossing over between duplicated IS elements in the genome.
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The transposition of an IS element requires an enzyme encoded by the IS element called transposase. The transposase recognizes the IR sequences of the element to initiate transposition.
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Transposons (Tn) contains genes for the insertion of the DNA segment into the chromosome and mobilization of the element to other locations on the chromosome. – there are two types of bacterial transposons, composite and noncomposite.
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Composite transposons – exemplified by Tn10, are complex transposons with a central region containing genes, flanked on both sides by IS elements – they may be thousands of base pairs long
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Noncomposite transposons – exemplified by Tn3, also contain genes such as those conferring resistance to antibiotics, but they do not terminate with IS elements. However
at their ends they have inverted repeated sequences that are required for transposition. Enzymes for transposition are encoded by genes in the central region of noncomposite transposons. Transposase catalyzes the insertion of a transposons into new sites, and resolvase is an enzyme involved in the particular recombinational events associated with transposition. -
Cointegration mechanism – used for the transposition of a transposon from one DNA to another (eg. From a plasmid to a bacterial chromosome)
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First the donor DNA containing the transposable element fuses with the recipient DNA to form a co-integrate. Because of the way this occurs, the transposable element is duplicated and one copy is located at each junction between the donor and the recipient. Next, recombination between the duplicated transposable elements resolves the cointegrate into two genomes, each with one copy of the element. Because the transposable element becomes duplicated, the process is called replicative transposition
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a second type of transposition mechanism involves the movement of a transposable element from one location to another on the same or different DNA without replication of the element. – this is called conservative (nonreplicative) transposition) (ie. the element is lost from the original position when it transposes)
Transposable Elements in Eukaryotes -
functional eukaryotic transposable elements have genes that encode enzymes required for transposition, and they can integrate into chromosomes at a number of sites. Thus, such elements can effect the function of any gene., effects range from activation to repression of adjacent genes to chromosome mutations such as duplications, deletions, inversions, translocations, or breakage. (typically results in a null mutation)
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Several families of transposable elements – each family has two forms: autonomous which can transpose themselves, and nonautonomous which cannot transpose by themselves because they lack the gene transposition
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When an autonomous element is inserted into a host gene, the resulting mutant allele is unstable, because the element can excise and transpose to a new location. This transposition out of a fene is higher than the spontaneous reversion frequency for a regular point mutation; therefore, the allele produced by an autonomous element is called a mutable allele.
SECTION ON CORN GOES HERE (IS MISSING) KEYNOTE: the transposition mechanism of plant transposable elements is similar to that of bacterial IS elements or transposons. Transposable elements integrate at a target site by a precise mechanism, so that the integrated elements are flanked at the insertion site by a short
duplication of target-site DNA of a characteristic length. Many plant transposable elements occur in families, the autonomous elements of which are able to transpose only when activated by an autonomous element in the same genome. Most autonomous elements are derived from autonomous elements by internal deletions or complex sequence rearrangement. Human Retrotransposons -
LINEs (long interspersed sequences) - are repeated sequences, 1000-7000 bps long, interspersed with unique sequence DNA.
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SINEs (short interspersed sequences) - are 100-400 bps long
**these apply to the human genome**
Chromosomal mutations – changes involving whole chromosomes or sections of them
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Point mutation – a change of one or a few base pairs – may change the phenotype of the organism if it occurs within the coding region of a gene or in the sequences regulating the gene
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Gene mutations – affect gene function - can alter a phenotype by changing the function of the protein
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Transposable elements – genetic change occurring when certain mobile genetic elements in chromosome move from one location to another in the genome
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Transposition – change in position
DNA Mutation Adaptation versus Mutation -
Adaptation – the environment induced an adaptive inheritable change
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Some geneticists thought that variation among organisms resulted from random mutations that sometimes happened to be adaptive
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Others thought that variations resulted from adaptation; that is, the environment induced an adaptive inheritable change
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The Adaptation Theory – Lamarckism – the doctrine of the inheritance of acquired characteristics
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Keynote: heritable adaptive traits result from random mutation, rather than by adaptation as a result of induction by environmental influences
Mutations Defined -
Mutation – is the process by which the sequence of base pairs in a DNA molecule is altered – may result in a change to either a DNA base pair or a chromosome
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Somatic mutation – (in multicellular organisms) the mutant characteristic affects only the individual in which the mutation occurs and it not passed on to the succeeding generation
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Germ-line mutation – may be transmitted by the gametes to the next generation, producing an individual with the mutation in both its somatic and its germ-line cells
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Mutation rate – probability of a particular kind of mutation as a function of time, such as the number of mutations per nucleotide pair per generation
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Mutation frequency – the number of occurrences of a particular kind of mutation, expressed as the proportion of cells or individuals in a population; such as the number of mutations per 100,000 organisms
Types of Point Mutations -
Base-pair substitutions – a change from one base pair to another in DNA. Depending on how the gene is translated to amino acid sequences it can result in no change to the protein, an insignificant change or a noticeable change. Two types:
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(1) a transition mutation – from one purine-pyrimidine base pair to the other purinepyrimidine base pair, such as A-T to G-C; this means that the purine on one strand of the DNA is changed to the other purine, while the pyrimidine ion the complementary strand is changed to the other pyrimidine
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(2) a transversion mutation – a mutation from a purine-pyrimidine base pair to a pyrimidine-purine base pair, such as G-C to C-G or A-T to C-G
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Missense mutation – is a gene mutation in which a base-pair change causes a change in an mRNA codon so that a different amino acid is inserted into the polypeptide – a phenotypic change may or may not result, depending on the amino acid change involved
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Nonsense mutation – is a gene mutation in which a base-pair change alters an mRNA codon for an amino acid to a stop (nonsense) codon (UAG, UGA, UAA) – this causes premature termination of polypeptide synthesis, so shorter than normal polypeptide fragments (often non-functional) are released from the ribosomes
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Neutral mutation – is a base pair change in a gene that changes a codon in the mRNA such that the resulting amino acid substitution produces no detectable change in the function of the protein translated from that message – a neutral mutation is a subset of missense mutations in which the new codon codes for a different amino acid that is chemically equivalent to the original or the amino acid is not functionally important and therefore does not affect the proteins function
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Silent mutation - also known as a synonymous mutation – is a mutation that changes a base pair in a gene, but the altered codon in the mRNA specifies the same amino acid in the protein – in this case the protein obviously has wild type function – silent mutations most often occur by changes such as this at the third-wobble-position of a codon
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Frameshift mutation – usually results in a non-functional protein – if one or more base pairs are added or deleted from a protein-coding gene, the reading frame of mRNA can change downstream of the mutation – therefore incorrect amino acids are added to the polypeptide chain after the mutation – these mutations may generate new stop codons, resulting in a shortened or lengthened polypeptide chains
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Keynote: mutation is the process by which the sequence of base pairs in a DNA molecule is altered. Mutations that affect a single base pair of DNA are called base-pair substitutions mutations. Base-pair substitutions and single base-pair insertions or deletions are called point shift mutations. Mutations in the sequences of genes are called gene mutations
Reverse Mutations and Suppressor Mutations -
Point Mutations are divided into two classes based on how they affect phenoty[es:
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(1) a forward mutation – changes a wild type phenotype gene to a mutant gene
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(2) a reverse mutation – also known as a reversion or back mutation – changes a mutant gene at the same site so that it functions in a completely wild-type or nearly wild type way
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Reversion of a nonsense mutation occurs when a base pair change results in a change of mRNA nonsense codon to a codon for an amino acid. if this reversion is back to the wild type amino acid, the mutation is a true reversion – if the reversion is to some other amino acid, the mutation is a partial reversion, and complete or partial may be restored, dpending on the change
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Suppressor mutation – a mutation at a different site from that the original mutation. A suppressor mutation masks or compensates for the effects of the initial mutation, but does not reverse the original mutation
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Intragenic (in the same gene but different site) and intergenic (in a different gene from the original) suppressors – operate to decrease or eliminate deleterious effects of the original mutation.
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Intragenic suppressors – act by altering a different nucleotide in the same codon where the original mutation occurred or by altering a nucleotide in a different codon
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Intergenic suppression is the result of a second mutation in another gene. Genes that cause the suppression of mutations are called suppressor genes.
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Keynote: reverse mutations occur at the same site as the original mutation and cause the genotype to change from mutant to wild type. A suppressor mutation is one that occurs at a second site and completely or partially restores a function that was lost or altered because of a primary mutation. Intragenic suppressors are suppressor mutations that occur within the same gene where the original mutation occurred, but at a different site. Intergenic suppressors are suppressor mutations that occur in a suppressor gene – a gene different from the one with the original mutation.
Spontaneous and Induced Mutations -
Mutagenesis – the creation of mutations, can occur spontaneously or can be induced.
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Spontaneous mutations – are naturally occurring mutations
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Induced mutations – occur when an organism is exposed to either deliberately or accidental to a physical or chemical agent, known as a mutagen, that interacts with DNA to cause a mutation
Spontaneous Mutations -
All types of point mutations occur spontaneously
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They can occur during DNA replication, as well as during other stages of cell growth or division
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They can also result from the movement of transposable genetic elements
DNA Replication Errors -
Tautomers – when a base chemically can exist in alternative states – when a base changes its state it has undergone tautomeric shift – in DNA the keto form of each base is responsible for the normal Watson-Crick base pairing of T with A and C with G – however non-Watson-Crick base pairing can result in a base is in a rare tautomeric state, the enol form.
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Small additions and deletions can occur spontaneously during replication, they occur because of displacement-looping out- of bases from either the template or the growing DNA strand, generally in regions where a run of the same base or of a repetitive sequence is present. If DNA loops out from the template strand, DNA polymerase skips the looped-out base or bases, producing a deletion mutation; if DNA polymerase synthesizes an untemplated base or bases, the new DNA loops out from the template, producing an addition. An addition or deletion mutation un the coding region of a structural gene is a frameshift mutation if it involves other that 3 bp or a multiple of 3 bp
Spontaneous Chemical Changes -
Depurination – the loss of a purine from the DNA when the bond hydrolyzes between the base and the deoxyribose sugar, resulting in an apurinic site. – it occurs because the covalent bond between the sugar and the purine is much less stable than the bond between the sugar and the pyrimidine and is prone to breakage
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If such lesions are not repaired, there is no base to specify a complementary base during DNA replication, and the DNA polymerase may stall or dissociate from the DNA
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Deamination – is the removal of an amino group from a base – for example the deamination of cytosine produces uracil
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A repair system replaces most of the uracils in DNA thereby minimizing mutational consequences of cytosine deamination
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However if uracil is not replaces an adenine will be incorporated into the new DNA strand opposite it during replication, eventually resulting in a CG to TA transition mutation.
Induced Mutations -
Mutations can be induced by exposing organisms to physical mutagens, such as radiation, or to chemical mutagens. Deliberately induced mutations have played an important role in the study of mutations. Since the rate of spontaneous mutation is so low, mutagens are used to increase the frequency of a mutation so that a significant number of organisms have mutations in the gene being studied.
Radiation -
Radiation occurs in non-ionizing or ionizing forms. Ionization occurs when energy is sufficient to knock an electron out of an atomic shell and hence break covalent bonds. Except for UV, non-ionizing radiation does not induce mutations; but all forms of ionizing radiation such as X-rays, cosmic rays and radon can induce mutations
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UV light causes mutations by increasing the chemical energy of certain molecules, such as pyrmidines, in DNA. One effect of UV radiation on DNA is the formation of abnormal chemical bonds between adjacent pyrimidine molecules in the same strand of the double helix. This bonding is induced mostly between adjacent thymines, forming what are called thymine dimers (TT, CC, CT, TC). These unusual pairings produce a bulge in the DNA strand and disrupts the normal pairing of T with A. Replication cannot proceed past the lesion so the cell will die if enough pyrimidine dimmers remain unrepaired.
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Further (clarify) – ionizing radiation penetrates tissues, colliding with molecules and knocking electrons out of orbits, thereby creating ions. The ions can result in the breakage of covalent bonds, including those in the sugar-phosphate backbone of DNA.
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High dosages of ionizing radiation kills cells – hence their use in treating some forms of cancer. At certain low levels of ionizing radiation, point mutations are commonly produced; at these levels, there is a linear relationship between the rate of point mutations and the radiation dosage
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X-rays, Radon and Uranium are all sources of radiation and can all induce mutations
Chemical Mutagens -
Chemical mutagens include both naturally occurring chemicals and synthetic substances
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3 groups based on their mechanisms of action: base analogs, base-modifying analogs and intercalating agents
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Base analogs and intercalating agents depend on replication
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Base modifying agents can induce mutations at any point of the cell cycle
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Base analogs – are bases that are similar to those normally found in DNA. Like normal bases, base analogs exist in normal and rare tautomeric states. In each of the two states, the base analog pairs with a different base in DNA. Because base analogs are so similar to the normal nitrogen bases, they may be incorporated into DNA in place of the normal bases.
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An example of a base analog: 5-bromouracil (5 BU), which has a bromine residue instead of the methyl group of thymine. In its normal state 5 BU resembles thymine and pairs with adenine in DNA. In its rare state, it pairs with guanine. 5 BU induces mutations by switching between its two chemical states once the base analog has been incorporated into the DNA. If 5 BU is incorporated in its normal state, it pairs with adenine. If it then changes into its rare state during replication, it pairs with guanine instead. In the next round of replication, the 5 BU-G base pair is resolved into a C-G base pair instead of the T-A base pair. By this process a transition mutation is produced. 5 BU can also induce a CG-to-TA transition mutation if it is first incorporated into DNA in its rare state and then switches to the normal state during replication. Thus, 5 BUinduced mutations can be reverted by a second treatment of 5 BU.
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Base-modifying agents – are chemicals that act as mutagens by modifying the chemical structure and properties of bases. There’s three types of mutagens that work in this way: a deaminating agent, a hydroxylating agent, and an alkylating agent o
Nitrous acid, HNO2 is a deaminating agent that removes amino groups (-NH2) from the bases G, C and A. Treatment of guanine with nitrous acid produces xanthine, but because this purine base has the same pairing properties as guanine, no mutation results. Treatment of cytosine with nitrous acid produces uracil, which pairs with adenine to produce a CG-to-TA transition mutation during replication. Likewise, nitrous acid modifies adenine to produce hypoxanthine, a base that pairs with cytosine rather than thymine, which results in an AT-GC transition mutation. Therefore, a nitrous acid-induced mutation can be reverted by a second treatment with nitrous acid.
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Hydroxylamine (NH2OH) Is a hydroxylating mutagen that reacts specifically with cytosine, modifying it by adding a hydroxyl group (OH) so that it pairs with adenine instead of guanine. Mutations induced by hydroxylamine can only be CG-to-TA transitions, so hydroxylamine-induced mutations cannot be reverted by a second treatment with this chemical =. However, they can be reverted by treatment with other mutagens (such as 5 BU and nitrous acid) that cause TA-toCG transition mutations.
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Methylmethane sulfonate (MMS) is one of a diverse group of alkylating agents that introduce alkyl groups (-CH3, -CH2, CH3) onto the bases at a number of locations. Most mutations caused by alkylating agents result from the addition of an alkyl group to the 6-oxygen of guanine to produce O6-methylguanine. They methylated guanine pairs with thymine rather than cytosine, giving GC-to-AT transitions.
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Intercalating agents – such as proflavin, acridine and ethidium bromide (commonly used to stain DNA in gel electrophoresis experiments)- insert (intercalate) themselves between adjacent bases in one or both strands of the DNA double helix causing the helix to relax. If the intercalating agent inserts itself between adjacent base pairs of the DNA strand that is the template for new DNA synthesis, an extra base (chosen at random) is inserted into the new DNA strand opposite the intercalating agent. After one more round of replication, during which the intercalating agent is lost, the overall result is a base pair addition mutation. If the intercalating agent inserts itself into the new DNA strand in place of a base, then when that DNA double helix replicates after the intercalating agent is lost, the result is a base-pair deletion mutation. – if a base-pair addition or deletion point mutation occurs in a protein-coding gene, the result is a Frameshift mutation.
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KEYNOTE: mutations can be produced by exposure to chemical mutagens. If the genetic damage caused by the mutagen is not repaired, mutations result. Chemical mutagens act in a variety of ways, such as by substituting for normal bases during DNA replication, modifying the bases chemically, and intercalating themselves between adjacent bases during replication.
Site-specific in Vitro Mutagenesis of DNA -
Site-specific mutagenesis – mutating a gene at specific positions in the base-pair sequence in the test tube and then introduce the mutated gene back into the cell and investigate the phenotypic changes produced by the mutation in vivo.
Environmental Mutagens -
Our exposure to chemicals occurs primarily through eating food, absorption through the skin, and inhalation.
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For a mutagenic chemical to cause DNA changes it must enter cells and penetrate the nucleus, which many chemicals cannot do.
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Some chemicals are converted from mutagenic to non-mutagenic by our metabolism.
The Ames Test: A Screen for Potential Mutagens -
Carcinogens – chemical agents that induce mutations that result in tumorous or cancerous growth
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The mutations are typically base-pair substitutions that produce missense or nonsense mutations, or base pair additions or deletions that produce Frameshift mutations
The Ames test – assays the ability of chemicals to revert mutant strains of the bacterium Salmonella typhimurium to the wild type -
The Ames test, approximately 108 cells of tester bacteria that are auxotrophic for histidine (his mutants) are spread with or without mixture of rat, mouse, or hamster liver enzymes on a culture plate lacking histidine. Histidine (his) auxotrophs require histidine
in the growth medium in order to grow; normal (his+) individuals do not. An array of tester bacterial strains are available that allow detection of base-pair substitution mutations and frameshift mutations in the test. The liver enzymes called the S9 extract, are used because, many chemicals are not mutagenic themselves but are metabolized to mutagens (and carcinogens) in the body, often in the liver and other tissues. -
Test chemical is placed on the plate which is incubated over night and examined for colony formation the following day – a positive result in the Ames test is a significantly higher number of revertants near the test chemical disk than is seen on the control plate.
Detecting Mutations -
Mutations of haploid organisms are readily detectable because there is only one copy of the genome.
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Dominant mutations are also readily detectable, and X-linked recessive mutations can be detected because they are expressed in half of the sons of a mutated, heterozygous female. However, autosomal recessive mutations can be detected only if the mutation is homozygous.
Visible Mutants -
Visible mutants – affect the morphology or physical appearance of an organism. Examples of mutants are eye color, wing shape mutants, coat-color (like in albinos), colony size mutants of yeast, and plaque morphology mutants of bacteriophages.
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Since visible mutants are readily apparent, screening is done by inspection
Nutritional Mutants -
An auxotrophic (nutritional) mutant is unable to make a particular molecule essential for growth
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Auxotrophic mutants are most readily detected in microorganisms such as E. coli and yeast that grow on simple defined growth media from which they synthesize the molecules essential to their growth. A number of selection and screening procedures are available to isolate auxotrophic mutants.
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Replica plating – can be used to screen for auxotrophic mutants of any microorganism that grows in discrete colonies on a solid medium – samples from a culture of mutagenized or an unmutangenized colony-forming organism or cell type are plated onto a medium containing the nutrients appropriate for the mutants desired.
Conditional Mutants -
Conditional mutants – reduce the activity of gene products only under certain conditions – a common type of conditional mutation is a temperature-sensitive mutant (in yeast) normally grow at 23, r not at all at 36 degrees in order to be isolated. – heat
sensitivity typically results from a missense mutation causing a change in the amino acid sequence of a protein so that, at the higher temperature, the protein assumes a nonfunctional shape Resistance Mutants -
Mutations can be induced for resistance to particular viruses, chemicals, or drugs.
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For example: in E. coli mutants resistant to phage T1 have been induced and some mutants are resistant to antibiotics
KEYNOYE: A number of screening procedures have been developed to isolate mutants of interest from a heterogeneous mixture of cells in a mutagenized population of cells. Repair of DNA Damage -
Mutagenesis involves damage to DNA
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Especially with high dosages of mutagens, the mutational damage can be considerable. What we see as mutations are DNA alterations that are not corrected by various DNA damage repair systems – (mutations = DNA damage – DNA repair)
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There are two general categories of repair systems based on the way they function: direct reversal repair systems correct damaged areas by reversing the damage, whereas excision repair systems cut out a damaged area and then repair the gap by new DNA synthesis.
Direct Reversal of DNA Damage -
Mismatch Repair by DNA Polymerase Proofreading
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When an incorrect nucleotide is inserted, the polymerase often detects the mismatched base pair and corrects the area by backspacing to remove the wrong nucleotide and the resuming synthesis in the forward direction.
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The mutator mutations in E. coli illustrate the importance of the 3’ to 5’ exonuclease activity of DNA polymerase fir maintaining a low mutation rate. Mutator mutants have a much higher than normal mutation frequency for all genes. These mutants have mutations in genes for proteins whose normal functions are required for accurate DNA replication.
Repair of UV-induced Pyrimi=dine Dimers -
Photoreactivation or light repair – UV light induced thymine (or other pyrimidine) dimmers are reverted directly to the original form by exposure to near UV-light in the wavelength range from 320 to 370 nm. Photoreactivation occurs when an enzyme called photolyase is activated by a photon of light and splits the dimmers apart. Strains with mutations in the phr gene are defective in light repair. Phtolyase has been found in bacteria and in simple eukaryotes but not in humans,
Repair of Alkylation Damage -
Alkylating agents transfer alkyl groups (usually methl or ethyl groups) onto the bases. The mutagen MMS methylates the oxygen of carbon-6 in guanine. The enzyme removes the methyl group from the guanine, thereby chaning the base back to its original form. A similar specific system exists to repair alkylated thymine. Mutations of the genes encoding these repair enzymes result in a much higher rate of spontaneous mutations.
Excision Repair of DNA Damage -
Many mutations affect only one of the two strands. In such cases, the DNA damage can be excised and the normal strand used as a template for producing a corrected strand. Depending on the damage, excision may involve a single base or nucleotide, or two or more nucleotides. Each excision repair system involves a mechanism to recognize the specific DNA damage it repairs.
Base Excision Repair -
Damaged single bases or nucleotides are most commonly repaired by removing the base ot the nucleotide involved and the inserting the correct base or nucleotide
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Base excision repair – a repair glycosylase enzyme removes the damaged base from the DNA by cleaving the bond between the base and the deoxyribose sugar. Other enzymes then cleave the sugar-phosphate backbone before and after the now baseless sugar, releasing the sugar and leaving the gap in the DNA chain. The gap is filled with the correct nucleotide by a repair DNA polymerase and DNA ligase, with the opposite DNA strand used as the template. Mutations caused by depurination or deamination are examples of damage that may be repaired by base excision repair.
Nucleotide Excision Repair -
Dark repair or excision repair system (nucleotide excision repair – NER) – because the normal photoreactive repair system cannot operate n the dark, investigators hypothesized that there must be another light-independent repair system.
Methyl-Directed Mismatch Repair -
Methyl-Directed Mismatch Repair – correcting mismatched base pairs left after DNA replication – the system recognizes the incorrect bases, and then carries out repair synthesis
Translesion DNA Synthesis and the SOS Response -
Lesions that block the replication machinery from proceeding past that point can be lethal if unrepaired
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Translesion DNA synthesis – allows replication to continue past the lesions. The process involves a special class of DNA polymerases that are synthesized only in response to
DNA damage. In E. coli such DNA damage activates a complex system called SOS response (called SOS because it is a last resort, emergency response to mutational damage). The SOS response allows the cell to survive otherwise lethal events, although often at the expense of generating new mutations. KEYNOTE: Mutations constitute damage to the DNA. Both prokaryotes and eukaryotes have a number of repair systems that deal with different kinds of DNA damage. All the systems use enzymes to make the correction. Without such repair systems, lesions would accumulate and be lethal to the cell or organism. Not all lesions are repaired, and mutations do appear, but at low frequencies. At high doses of mutagens, repair systems are unable to correct all of the damage, and cell death may result. Human Genetic Diseases Resulting from DNA Replication and Repair Mutations -
Some human genetic diseases are attributed to defects in DNA replication or repair
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Example: xeroderma pigmentosum (OMIM 278700) is caused by homozygosity for a recessive mutation in a repair gene. Individuals with this lethal affliction are photosensitive, and portions of their skin that have been exposed to light show intense pigmentation, freckling and warty growths that can become malignant.
Transposable Elements General Features of Transposable Elements -
Transposable elements are normal, ubiquitous components of the genomes of prokaryotes and eukaryotes. Transposable elements fall into two general classes based on how they move from location to location in the genome. One class- found in both prokaryotes and eukaryotes – moves as a DNA segment. Members of the other class – found only in eukaryotes – are related to retroviruses and move via an RNA. First an RNA copy is synthesized; then a DNA copy of that RNA is made, and it integrates at a new site in the genome.
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In bacteria, transposable elements can move to new positions on the same chromosome (because there is only one chromosome) or onto plasmids or phage chromosomes
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In Eukaryotes, transposable elements may move to new positions within the same chromosome or to a different chromosome.
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In both bacteria and eukaryotes, transposable elements insert new chromosome locations with which they have no sequence homology; therefore, transposition is a process different from homologous recombination (recombination between matching DNA sequences) and is called nonhomologous recombination.
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Transposable elements are important due to the genetic changes they cause – for example, they can produce mutations by inserting into gene regulatory sequences (such as disrupting promotes on the element), and they can produce various kinds of chromosomal mutations through the mechanics of transposition.
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The frequency of transposition, though typically low, varies with the particular element, if the frequency were high, the genetic changes caused by the transpositions would likely kill the cell
Transposable Elements in Bacteria -
Two examples of transposable elements in bacteria are insertion sequence(IS) elements and transposons (Tn)
Insertion Sequences -
Insertion Sequences or IS element, is the simplest transposable element found in bacteria. An IS element contains only genes required to mobilize the element and insert it into a new location in the genome. IS elements are normal constituents of bacterial chromosomes and plasmids.
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IS elements were first identified in E. coli as a result of their effects on the expression of three genes that control the metabolism of the sugar galactose. Some mutations affecting the expression of these genes did not have properties typical of point mutations or deletions, but rather had an insertion of approx 800 bps DNA segment into a gene. This particular DNA segment is now called insertion sequence 1, or IS1, and the insertion of IS1 into the genome is an example of a transposition event.
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When IS elements integrate at random points along the chromosome, they often cause mutations by disrupting either the coding sequence of a gene or a gene’s regulatory region. Promoters within IS elements themselves may also have effects by altering the expression of nearby genes.
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In addition, the presence of an IS element in the chromosome can cause mutations such as deletions and inversions in the adjacent DNA
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Finally deletion and insertion events can also occur as a result of crossing over between duplicated IS elements in the genome.
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The transposition of an IS element requires an enzyme encoded by the IS element called transposase. The transposase recognizes the IR sequences of the element to initiate transposition.
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Transposons (Tn) contains genes for the insertion of the DNA segment into the chromosome and mobilization of the element to other locations on the chromosome. – there are two types of bacterial transposons, composite and noncomposite.
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Composite transposons – exemplified by Tn10, are complex transposons with a central region containing genes, flanked on both sides by IS elements – they may be thousands of base pairs long
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Noncomposite transposons – exemplified by Tn3, also contain genes such as those conferring resistance to antibiotics, but they do not terminate with IS elements. However
at their ends they have inverted repeated sequences that are required for transposition. Enzymes for transposition are encoded by genes in the central region of noncomposite transposons. Transposase catalyzes the insertion of a transposons into new sites, and resolvase is an enzyme involved in the particular recombinational events associated with transposition. -
Cointegration mechanism – used for the transposition of a transposon from one DNA to another (eg. From a plasmid to a bacterial chromosome)
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First the donor DNA containing the transposable element fuses with the recipient DNA to form a co-integrate. Because of the way this occurs, the transposable element is duplicated and one copy is located at each junction between the donor and the recipient. Next, recombination between the duplicated transposable elements resolves the cointegrate into two genomes, each with one copy of the element. Because the transposable element becomes duplicated, the process is called replicative transposition
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a second type of transposition mechanism involves the movement of a transposable element from one location to another on the same or different DNA without replication of the element. – this is called conservative (nonreplicative) transposition) (ie. the element is lost from the original position when it transposes)
Transposable Elements in Eukaryotes -
functional eukaryotic transposable elements have genes that encode enzymes required for transposition, and they can integrate into chromosomes at a number of sites. Thus, such elements can effect the function of any gene., effects range from activation to repression of adjacent genes to chromosome mutations such as duplications, deletions, inversions, translocations, or breakage. (typically results in a null mutation)
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Several families of transposable elements – each family has two forms: autonomous which can transpose themselves, and nonautonomous which cannot transpose by themselves because they lack the gene transposition
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When an autonomous element is inserted into a host gene, the resulting mutant allele is unstable, because the element can excise and transpose to a new location. This transposition out of a fene is higher than the spontaneous reversion frequency for a regular point mutation; therefore, the allele produced by an autonomous element is called a mutable allele.
SECTION ON CORN GOES HERE (IS MISSING) KEYNOTE: the transposition mechanism of plant transposable elements is similar to that of bacterial IS elements or transposons. Transposable elements integrate at a target site by a precise mechanism, so that the integrated elements are flanked at the insertion site by a short
duplication of target-site DNA of a characteristic length. Many plant transposable elements occur in families, the autonomous elements of which are able to transpose only when activated by an autonomous element in the same genome. Most autonomous elements are derived from autonomous elements by internal deletions or complex sequence rearrangement. Human Retrotransposons -
LINEs (long interspersed sequences) - are repeated sequences, 1000-7000 bps long, interspersed with unique sequence DNA.
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SINEs (short interspersed sequences) - are 100-400 bps long
**these apply to the human genome**
