Bio3400 Chapter 15 Gene Mutation, DNA Repair, and Transposition
  1. A mutation is a         in DNA sequence and can occur spontaneously or be          by an external agent (mutagen).

      Point mutations are base substitutions in which one base pair is altered. Insertions or deletions can lead to frameshift mutations where all subsequent downstream amino acids are changed. Frameshifts involving multiples of three nucleotides can restore the initial reading frame.

      Spontaneous mutation rates range from 1/100,000 per gamete in mice to 1/100 million per replication in bacteria and viruses. The variation may reflect the relative efficiency of their DNA proofreading and repair systems.
  2.             shifts in nucleotides can result in             mutations due to anomalous base pairing.

      Nucleotides can exist in tautomeric forms (structural isomers) by a position change of a proton (tautomeric shift). The less common, transient tautomers can form hydrogen bonds with noncomplementary bases. The anomalous pairing is always between a pyrimidine and a purine, as shown in the T - G and C - A pairs.

      Nitrogenous bases can be either purines ot pyrimidines; each atom in the ring is assigned a number. The double-ring purines are adenine (A) and guanine (G). The single-ring pyrimidines are cytosine (C), thymine (T), and uracil (U).
      Both DNA and RNA contain A, C, and G; only DNA contains T, while only RNA contains U. 10_09-nitrogenous_bases.jpg,860,450-->

      Transition mutation. A rare tautomer in the template strand pairs with a noncomplementary base during DNA replication. In this example, a mormal T - A pair forms an abnormal C - A pair in one daughter helix. next

      In the next round of replication, the mismatched members of the base pair separate; the tautomer usually shifts back to its normal isomer. Replication of the 2 strands by normal base pairing results in a point mutation called a transition mutation, where a purine substitutes for a purine, or a pyrimidine substitutes for a pyrimidine. In this example, the wild type T - A pair has mutated to C - G. 15_03-transition_mutation2.jpg,646,600-->
  3. DNA base damage by              can also lead to mutation.

      Chemical mutagens such as nitrous acid (HNO[2]) may cause nucleotide deamination: conversion of an amino group to a keto group. Thus a transition mutation occurs: cytosine and adenine are converted to uracil (which base pairs with adenine) and hypoxanthine (which base pairs with cytosine), respectively.
  4. Other chemical mutagens include             agents which donate an alkyl group to nucleotides.

      Ethylmethane sulfonate (EMS, a mustard gas) is an alkylating agent that can donate an alkyl group (C[2]H[5]) to a keto group in guanine. The resulting 6-ethylguanine acts as an analog of adenine and pairs with thymine, leading to transition mutations.

      These sulfur-containing mustard gases were discovered during W.W. I. These alkylating agents also include EMS and EES. 15_T03-alkylating_agents.jpg,800,260-->
  5. In general, base          can lead to             mutations during DNA replication.

      5-bromouracil (5-BU) ia a derivative of uracil and behaves as a thymine analog, which increases the probability of a tautomeric shift from the normal keto form to the enol form, mis-pairing with guanine instead of adenine. After one round of replication, an A - T to G - C transition mutation results.
  6.           dyes cause             mutations by intercalating between purines and pyrimidines.

      Acridine dyes such as Proflavin and Acridine orange are about the same dimensions as nitrogenous base pairs and intercalate, or wedge, between the purines and pyrimidines of intact DNA. Intercalation introduces contortions in the DNA helix and causes deletions and insertions that create frameshift mutations.
  7. High energy radiation such as              (UV) light,    rays,        rays, and         rays are also mutagenic.

      Visible light is a form of radiation energy constituting part of the electromagnetic spectrum. Radiation with shorter wavelength than visible light, such as UV light, X rays, gamma rays, and cosmic rays, possesses more energy, and has a disruptive impact on organic molecules.

      The short wave radiation such as X rays are called ioning radiation. They penetrate deep into tissues, causing ionization (loss of electrons) of molecules which become reactive free radicals which can cause physical damage to DNA. Exposure to ionizing radiation has been shown to be directly related to the induction of mutations. 15_10-X-ray_mutations.jpg,696,568-->

      Exposure to UV radiation can result in the creation of thymine dimers in DNA. The dimers distort the DNA conformation and result in higher error rates during DNA replication. If the erros are not corrected, they can lead to tumors.

      Nucleotide excision repair.
    • Damaged DNA is recognized by uvr (ultraviolet repair) proteins.
    • A number of nucleotides is clipped out around the lesion by a nuclease.
    • The gap is filled by DNA polymerase I and DNA ligase
    • The error is corrected. 15_17-nucleotide_excision_repair.jpg,276,500-->

      The recessive genetic disorder xeroderma pigmentosum results in defective NER and cannot repair the thymine dimers from UV damage. The 4-year-old boy shows marked skin lesions, including two cancers on his nose. The 18-year-old girl on the right has been carefully protected from sunlight since her diagnosis of XP in infancy; several cancers have been removed. 15_18-xeroderma_pigmentosum.jpg,532,600-->
  8.                repeat sequences can cause several human genetic disorders.

      Abnormal amounts of trinucleotide repeat sequences can cause human genetic disorders such as fragile X syndrome, Huntington disease, myotonic dystrophy, and spinobulbar muscular atrophy. The number of repeats may increase in each generation, resulting in genetic anticipation: earlier age of onset and more severe of symptoms.
  9. The       test uses bacteria selected for their sensitivity to specific types of mutagenesis to screen compounds for potential mutagenicity.

      The Ames test exposes auxotrophic strains of Salmonella (his^) to a chemical compound to assess its mutagenicity. Liver extract is added, since metabolic modification of these compounds in the liver may make them mutagenic. The mutagenicity of a chemical is reflected in the frequency of reverse mutations, which yields wild-type (his^+) prototrophs.
  10.                  repair can restore gaps created during DNA replication due to damaged nucleotides.

      Postreplication repair. DNA danage (such as a thymine dimer) may cause a gap on the newly synthesized strand during replication. step 2 step 3

      Postreplication repair: 2. The E. coli RecA protein catalyzes a recombination from the other, undamaged parental strand. next 14_F11_02-postreplication_repair.jpg,556,460-->

      Postreplication repair: 3. The new gap is filled by DNA polymerase and DNA ligase. This postreplication repair is also referred to as homologous recombination repair. 14_F11_03-postreplication_repair.jpg,560,460-->

      These DNA polymerases cannot initiate DNA synthesis, but can elongate an existing DNA or RNA strand (primer). All three possess 3' to 5' exonuclease activity which allows proofreading to correct mismatched base pairs. DNA polymerase I fills gaps in the DNA and also removes the primer by its 5' to 3' exonuclease activity. Polymerases II is involved in DNA repair. Polymerase III is the main enzyme for 5' to 3' polymerization in vivo.

      The active form of DNA polymerase III is called a holoenzyme, a dimer with 10 different polypeptide subunits. The a (alpha), e (epsilon) and th (theta) subunits make up the core enzyme to perform polymerization and proofreading. The g (gamma) complex is involved in "loading" the enzyme onto the template at the replication fork. The b (beta) subunit serves as a "clamp" and prevents the core enzyme from falling off the template during polymerization. The t (tau) subunit functions to dimerize two core polymerases allowing simultaneous synthesis of both strands of the helix. The holoenzyme and several other proteins at the replication fork together form a huge complex called the replisome. 11_T03-DNA_polymerase_III-holoenzyme.jpg,648,564-->
  11.                    repair in prokaryotes can remove thymine dimers caused by UV light.

      Photoreactivation repair. In E. coli, the photoreactivation enzyme (PRE) can cleave the bonds between thymine dimers, energized by a photon of blue light. The reaction reverses the effect of UV radiation on DNA.
  12. Two types of excision repair can also repair damaged DNA:       excision repair (BER) and             excision repair (NER).

      Base excision repair in E. coli.
      1. A base pair mismatch is recognized by DNA glycosylase, which removes the base from the sugar, creating an apurinic/apyrimidinic (AP) site.
    • AP endonuclease makes a cut in the phosphodiester backbone at the AP site.
    • The gap is filled by DNA polymerase I and DNA ligase
    • The error is corrected.

      Nucleotide excision repair.
    • Damaged DNA is recognized by uvr (ultraviolet repair) proteins.
    • A number of nucleotides is clipped out around the lesion by a nuclease.
    • The gap is filled by DNA polymerase I and DNA ligase
    • The error is corrected.
  13.               elements (transposons or "jumping genes") can move within the genome.

        Barbara McClintock (Nobel 1983) studied pigment variation which can occur within a kernel of maize. This was controlled by two genes, Dissociation (Ds) and Activator (Ac), now known as transposable elements (transposons) that can move within the genome.

        Transposons. In absence of Ac (Activator), Ds (Dissociation) has no effect on expression of the color gene W, resulting in production of anthocyanin pigments and purple kernels. 15_22a-transposable_elements.jpg,800,116-->

        Transposons: 2. When Ac is present, Ds may be transposed to a region adjacent to W. Ds can induce chromosome breakage, leading to loss of function of the W gene.

        Transposons: 3. If Ds is transposed into the W gene, W gene is inhibited: no anthocyanin is produced. Subsequently, Ds can jump out of the W gene, and wild-type expression of W is restored. 15_22c-transposable_elements.jpg,800,508-->
    • The     (Activator) elements contain a              gene similar to that of bacterial            sequences and transposons.

      Transposons. The Ac sequence contains noncoding regions (Nc) and an open reading frame (ORF) which encodes a transposase enzyme and enables the sequence to "jump". The Nc also contains inverted terminal repeats not shown here. Ds elements are similar to Ac but contain a deletion within the transposase gene, making them dependent on the Ac element for transposition.

      Bacterial insertion sequence (IS) elements contain a gene that encodes an enzyme called transposase, which can make staggered cuts in DNA, like restriction enzymes. The ends of IS elements contain inverted terminal repeats (ITRs), which act as recognition sites for the binding of transposase.

      Cloning a recombinant gene. A vector, such as a virus or plasmid, and a DNA fragment produced by cutting with a restriction enzyme are joined to produce a recombinant DNA molecule that is transferred to a bacterial cell. The desired recombinant DNA is cloned into many copies by replication of the DNA and by division of the bacterial cell. 01_14-cloning.jpg,488,600-->

      Bacterial transposons (Tn elements) are larger than IS elements and contain protein-coding genes in addition to that for transposase. A transposon inserted into a plasmid contains inverted repeats (IR) that can form a heteroduplex after the original strands separate.

      The IR segments can reanneal after the original duplex has been separated, forming heteroduplex loops that can be seen under an electron microscope. 15_20-bacterial_transposon2.jpg,960,456-->