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Chapter 13 Mutation, DNA Repair,… © John Wiley & Sons, Inc.

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1 Chapter 13 Mutation, DNA Repair,… © John Wiley & Sons, Inc.

2 Amino acids in polypeptides are joined by what type of bond? a) Hydrogen b) no-Peptide c) Covalent d) Ionic e) Weak Which of the following is a secondary structure often observed in proteins? a)  helix b)  sheet c)  subunit d)  helix and  sheet e) All of these Which of the following is not a true statement about ribosomes? a) They are structured into large and small subunits b) They are comprised of RNA and protein c) The ribosomes in prokaryotes are typically larger than those found in eukaryotes d) The ribosome subunits associate during the initiation of translation e) All of these are false statements

3 Chapter Outline  Mutation: Source of the Genetic Variability Required for Evolution  The Molecular Basis of Mutation  Mutation: Basic Features of the Process  Mutation: Phenotypic Effects  Assigning Mutations to Genes by the Complementation Test  Screening Chemicals for Mutagenicity: The Ames Test  DNA Repair Mechanisms  Inherited Human Diseases with Defects in DNA Repair  DNA Recombination Mechanisms © John Wiley & Sons, Inc.

4 Mutation: Source of the Genetic Variability Required for Evolution © John Wiley & Sons, Inc.  Mutation --A change in the genetic material (molecular level)  Mutant  --an organism that exhibits a novel phenotype

5 Types of Mutations –Changes in chromosome number and structure –Point mutation--changes at specific nucleotide in a gene (A,T,C,G) –Insertion mutations--insert fragment of DNA –Deletion mutations--delete fragment of DNA © John Wiley & Sons, Inc.

6 “Virus”

7 Disruption of FGF -Receptor Function

8 Fibroblast Growth Factor Signaling is Essential for Mesoderm Production in Frog Embryos

9 Dominant mutations in human FGFR-3 (TM domain) Achondroplasia

10 Mutation and Evolution  Mutation is the source of all genetic variation (e.g.,chromatin remodeling).  Natural selection preserves the combinations best adapted (or NOT) to the existing environment. © John Wiley & Sons, Inc.

11 The Molecular Basis of Mutation Mutations alter the nucleotide sequences of genes in several ways, --the substitution of one base pair for another. (A for T) --the deletion (or addition) of one or a few base pairs. ( AT…….GC)

12 © John Wiley & Sons, Inc. Tautomeric Shifts: --chemical fluctuations, --conformation states (stable==========unstable) A:T C:G. Py Pu

13 Tautomeric Shifts Affect Base-Pairing © John Wiley & Sons, Inc. C:T T:G.

14 Mutation Caused by Tautomeric Shifts © John Wiley & Sons, Inc.

15 Base Substitutions  A transition replaces a pyrimidine with another pyrimidine or a purine for another purine.  A transversion replaces a pyrimidine with a purine or a purine with a pyrimidine. © John Wiley & Sons, Inc.

16 Frameshift Mutations: alteration of the open reading frame (ORF) © John Wiley & Sons, Inc.

17 Mutation Frequency  Frameshift, transition, transversion mutations are infrequent –Bacteria and phage: 10 –8 to 10 –10 per nucleotide pair per generation –Eukaryotes: 10 –7 to 10 –9 per nucleotide pair per generation © John Wiley & Sons, Inc. 1/10 7 to 1/10 9 Silent mutation: UCU=Ser; UCA, UCC, UCG = Ser

18 Induced Mutations  Induced mutations occur upon exposure to physical (energy) or chemical (reaction) mutagens.  Muller demonstrated that exposing Drosophila sperm to X-rays increased the mutation frequency.  Hermann J. Muller and Edgar Alternburg measured the frequency (>150 fold increase) of X-linked recessive lethal mutations in Drosophila. © John Wiley & Sons, Inc.

19 C: crossover suppressor l: recessive lethal mutation B: bar-eye mutation

20 The Electromagnetic Spectrum  X-rays induce mutations through ionization. (DNA ionization--radical anions and cations-- G. +)  Ultraviolet light induces mutations through excitation. © John Wiley & Sons, Inc.

21 Ionizing Radiation Causes Changes in Chromosome Structure  Ionizing radiation breaks chromosomes and can cause deletions, duplications, inversions, and translocations © John Wiley & Sons, Inc.

22 Stability of carbon-containing molecules

23 Mutagenesis by Ultraviolet Irradiation  Hydrolysis of cytosine to a hydrate may cause mis-pairing during replication  Cross-linking of adjacent thymine forms thymidine dimers, which block DNA replication and activate DNA repair mechanisms. © John Wiley & Sons, Inc.

24 UV-A 320 to 400 nm UV-B/C <300 nm

25 Types of Chemical Mutagens  Chemicals that are mutagenic to both replicating and non-replicating DNA (e.g., alkylating agents and nitrous acid)  Chemicals that are mutagenic only to replicating DNA (e.g., base analogs and acridine dyes) © John Wiley & Sons, Inc.

26 Chemical Mutagens © John Wiley & Sons, Inc.

27 Alkylating Agents  chemicals that donate alkyl groups to other molecules.  induce transitions, transversions, frameshifts, and chromosome aberrations (anomaly).  Alkylating agents of bases can change base-pairing properties. (GC to AT)  can also activate errors during repair processes. © John Wiley & Sons, Inc.

28 A Base Analog: 5-Bromouracil © John Wiley & Sons, Inc. --similar structures --incorporated into DNA --increase frequency of mis-pairing

29 Mutagenic Effects of 5-Bromouracil © John Wiley & Sons, Inc.

30 Nitrous Acid Causes Oxidative Deamination of Bases © John Wiley & Sons, Inc.

31 Intercalation of an Acridine Dye Causes Frameshift Mutations © John Wiley & Sons, Inc. --(+) charges molecules --Incorporated into DNA --DNA is more rigid --Change conformation (non-bending)

32 Hydroxylamine (NH 2 OH)  Hydroxylamine is a hydroxylating (OH) agent.  Hydroxylamine hydroxylates the amino group of cytosine and leads to G:C  A:T transitions. © John Wiley & Sons, Inc..

33 Mutations Induced by Transposons (Repeats) © John Wiley & Sons, Inc. Fragmets/segments of DNA that are capable to shift / translocate/move from one location to another

34 Expansion of Trinucleotide Repeats  Simple tandem repeats are repeated sequence of one to six nucleotide pairs (CGG, CAG and CTG).  Trinucleotide repeats can increase in copy number and cause inherited diseases (Fragile X Syndrome, Huntington disease, Spinocerebellar ataxia) © John Wiley & Sons, Inc.

35 Mutations are induced by chemicals, ionizing irradiation, ultraviolet light, and endo(exo)genous transposable genetic elements. Point mutations are of three types: (1)Transitions—purine for purine and pyrimidine for pyrimidine substitutions, (2)Transversions—purine for pyrimidine and pyrimidine for purine substitutions, and (3)Frameshift mutations—additions or deletions of one or two nucleotide pairs, which alter the reading frame of the gene distal to the site of the mutation. Chromatin remodeling==Epigenetics © John Wiley & Sons, Inc.

36 Mutation: Basic Features of the Process Mutations occur in all organisms from viruses to humans. They can occur spontaneously or be induced by mutagenic agents. Mutation is usually a random, non-adaptive process. © John Wiley & Sons, Inc.

37 Mutation: Somatic or Germinal  Germinal mutations occur in germ-line cells and will be transmitted through the gametes to the progeny.  Somatic mutations occur in somatic cells; the mutant phenotype will occur only in the descendants of that cell and will not be transmitted to the progeny. © John Wiley & Sons, Inc. puberty (10-14 years)

38 Mutation: Spontaneous or Induced  Spontaneous mutations occur without a known cause due to unknown agents in the environment.  Induced mutations result from exposure or organisms to mutagens, physical and chemical agents that cause changes in DNA, such as ionizing irradiation, ultraviolet light, or certain chemicals. © John Wiley & Sons, Inc.

39 Factors Influencing the Rate of Spontaneous Mutations  Accuracy of the DNA replication machinery  Efficiency of the mechanisms for the repair of damaged DNA  Degree of exposure to mutagenic agents in the environment © John Wiley & Sons, Inc.

40 Mutation: Usually a Random, Non- adaptive Process  Is mutation random (intrinsic) or directed by the environment?  Replica plating was used to identify the presence of antibiotic (chemical) resistant bacteria prior to treatment with an antibiotic (chemicals).  Environmental stress does not cause mutations but selects for mutants that are best adapted to the environmental stress. © John Wiley & Sons, Inc.


42 Mutation: A Reversible Process  Forward mutation—mutation of a wild- type allele to a mutant allele.  Reverse mutation (reversion)—a second mutation that restores the original phenotype. –Back mutation—a second mutation at the same site. –Suppressor mutation—a second mutation at a different location in the genome. © John Wiley & Sons, Inc.

43 Phenotype is not suppressed Phenotype is restored

44 Mutation: Phenotypic Effects The effects of mutations on phenotype range from no observable change to lethality. © John Wiley & Sons, Inc. DNA/RNA/ polypeptide

45 Types of Mutations  Isoalleles have no effect on phenotype or small effects that can be recognized only by special techniques.  Null alleles result in no gene product or totally non- functional gene products.  Recessive (Dominant) lethal mutations affect genes required for growth of the organisms and are lethal in the homozygous state. © John Wiley & Sons, Inc.

46 X-linked Recessive Lethal Mutations Alter the Sex Ratio © John Wiley & Sons, Inc. Monoploid: (Recessive or Dominant) positive mutational effect (phenotype) Diploid: (Recessive)--positive mutational effect (phenotype)--homozygous X-linked.....hemizygous

47 Recessive Mutations Often Block Metabolic Pathways (Recessive) lethal mutations Neutral mutations ( no effect on phenotype)

48 Expression of Wild-type and Mutant Alleles © John Wiley & Sons, Inc.

49 Mutations in Human Globin Genes  Adult hemoglobin (Hemoglobin A) contains two  chains and two  chains.  Hemoglobin in patients with sickle-cell anemia (Hemoglobin S) differs from Hemoglobin A at only one position.  The sixth amino acid in the  chain is glutamic acid in Hemoglobin A (HBB A ) and is valine in Hemoglobin S (HBB S ). This substitution is caused by mutation of a single base pair (T:A substitution). © John Wiley & Sons, Inc.

50 Tay-Sachs Disease  Tay-Sachs disease is an autosomal recessive disease.  The mutation causing Tay-Sachs disease is in the gene encoding hexosaminidase A. © John Wiley & Sons, Inc. not linked to sex chromosome

51 Conditional Lethal Mutations (Experimental)  Conditional lethal mutations are –Lethal in the restrictive condition but –Viable in the permissive condition.  Mutants with conditional lethal alleles can be propagated under the permissive condition, and the phenotype can be studied under restrictive condition. © John Wiley & Sons, Inc.

52 Conditional Lethal Mutants  Auxotrophs are unable to synthesize an essential metabolite that is synthesized by prototrophs. Auxotrophs can grow only when the essential metabolite is supplied in the medium.  Temperature-sensitive mutants will grow at one temperature but not at another.  Suppressor-sensitive mutants are viable only when a second genetic factor, a suppressor, is present. © John Wiley & Sons, Inc.

53 Morphogenesis in Bacteriophage T4  This pathway was identified using mutants, electron microscopy, and biochemistry.  Temperature-sensitive and suppressor-sensitive © John Wiley & Sons, Inc. # =gene


55 Assigning Mutations to genes by the Complementation test The complementation or trans test can be used to determine whether two mutations are located in the same chromosome or in two different chromosomes. © John Wiley & Sons, Inc. Genetic tool to demonstrate: One gene....One polypeptide

56 © John Wiley & Sons, Inc. Assigning Mutations Double heterozygote: Two mutations (m1 and m2) Wild-type (m1+ and m2+) Co-exists in one or two chromosomes coupling repulsion Arrangement (organized)

57 © John Wiley & Sons, Inc. Apr and W are recessive mutations X-linked mutations

58 © John Wiley & Sons, Inc.

59 (head) (tail)

60 © John Wiley & Sons, Inc. ?

61 Screening Chemicals for mutagenicity: The Ames test The Ames test provides a simple and inexpensive method for detecting the mutagenicity of chemicals (Carcinogens) -intracellular -extracellular -enviromental © John Wiley & Sons, Inc. Auxotrophic prototrophic

62 © John Wiley & Sons, Inc.

63 DNA Repair Mechanisms Living organisms contain many enzymes that scan their DNA for damage and initiate repair processes when damage is detected. © John Wiley & Sons, Inc.

64 DNA Repair Mechanisms in E. coli  Light-dependent repair (photo-reactivation).  Excision repair.  Mismatch repair.  Post-replication repair.  Error-prone repair system (SOS response). © John Wiley & Sons, Inc.

65 UV Light- Dependent Repair: Photolyase Cleaves Thymine Dimers. © John Wiley & Sons, Inc. --No endonuclease --No Poly --No ligase

66 Excision Repair (steps)  A DNA repair endonuclease or endonuclease-containing complex recognizes, binds to, and excised the damaged base or bases.  A DNA Polymerase fills in the gap, using the undamaged complementary strand of DNA as a template.  DNA ligase seals the break left by DNA polymerase. © John Wiley & Sons, Inc.

67 Types of Excision Repair  Base excision repair pathways remove abnormal or chemically modified bases.  Nucleotide excision repair pathways remove larger defects, such as thymine diners. © John Wiley & Sons, Inc.

68 Base Excision Repair © John Wiley & Sons, Inc. AP:apyrimidinic site

69 © John Wiley & Sons, Inc. ( )

70 Nucleotide Excision Repair © John Wiley & Sons, Inc.


72 Mismatch Repair in E. coli  Mismatching or mispairing of G and T (DNA polymerase/exonuclease proofreading activity)  The A in GATC sequences is methylated subsequent to DNA replication.  In newly replicated DNA, the parental strand is methylated, but the new strand is not. This difference allows the mismatch repair system to distinguish the new strand from the old strand.  The mismatched nucleotide is excised from the new strand and replaced with the correct nucleotide, using the methylated parental strand as a template. © John Wiley & Sons, Inc.

73 Exonuclease Endonuclease

74 Mismatch Repair in E. coli  MutS recognizes mismatches and binds to them to initiate the repair process.  MutH and MutL join the complex.  MutH cleaves the unmethylated strand at hemimethylated GATC sequences on either side of the mismatch.  Excision requires MutS, MutL, MutU (DNA helicase II), and an exonuclease.  DNA polymerase III fills in the gap, and DNA ligase seals the nick. © John Wiley & Sons, Inc. Me

75 Post-replication Repair in E. coli  A thymine dimer in the template strand blocks replication (DNA Polymerase III does not recognize thymidine dimer)  DNA Polymerase III restarts DNA synthesis past the dimer, leaving a gap in the nascent strand.  RecA binds to the single strand of DNA at the gap and mediates base pairing with the homologous segment of the sister double helix to fill the gap.  DNA polymerase fills the gap in the sister double helix, and DNA ligase seals the nick. © John Wiley & Sons, Inc.

76 The SOS Response in E. coli  If DNA is heavily damaged by mutagenic agents, the SOS response, which involves many DNA recombination, DNA repair, and DNA replication proteins, is activated.  DNA dependent DNA Polymerase V replicates DNA in damaged regions, but sequences in damaged regions cannot be replicated accurately.  This error-prone system eliminates gaps but increases the frequency of replication errors (Pol II, IV and V are low-fidelity polymerases) © John Wiley & Sons, Inc.

77 Induction of the SOS Response  In the absence of DNA damage, LexA binds to DNA regions that regulate transcription of SOS response genes and keeps their expression levels low.  When extensive DNA damage occurs, RecA binds to single-stranded regions of DNA in damaged regions.  This activates RecA, which stimulates LexA to inactivate itself. When LexA is inactivated, the SOS response genes are expressed. © John Wiley & Sons, Inc.

78 Inherited Human Diseases with Defects in DNA Repair Several inherited human disorders result from defects in DNA repair pathways. © John Wiley & Sons, Inc.

79 Xeroderma Pigmentosum (XP)  Individuals with XP are sensitive to sunlight (UV light).  The cells of individuals with XP are deficient in the repair of UV-induced damage to DNA.  Individuals with XP may develop skin cancer or neurological abnormalities. © John Wiley & Sons, Inc.


81 DNA Recombination Mechanisms Recombination between homologous DNA molecules involves the activity of numerous enzymes that 1-cleave, 2-unwind, 3-stimulate single-strand invasions of double helices (RecA proteins), 4-repair, and 5-join strands of DNA. © John Wiley & Sons, Inc.

82 Recombination  In eukaryotes, crossing over is associated with the formation of the synaptonemal complex during prophase of meiosis I (Chiasmata).  Crossing over involves the breakage of parental chromosomes and rejoining of the parts in new combinations.  The Holliday model and the double-strand break model are two explanations of the molecular basis of recombination. © John Wiley & Sons, Inc.

83 The Holliday Model: single strand break model

84 --DNA-dependent ATPase. --can hold a single strand and double strand together. --DNA synapsis reaction between a DNA double helix and a homologous region of single stranded DNA. --catalyzes branch migration begins.

85 The Recombination Intermediate is a Chi Structure © John Wiley & Sons, Inc.


87 5) Which of the following is considered a point mutation? 1. Substitution of the base A for the base C 2. Deletion of the base T 3. Insertion of the base G a) 1 b) 2 c) 3 d) 1 and 2 e) All of these Which base pair combinations can form when nitrogenous bases are present in their rare imino or enol states? a) A:T b) C:G c) A:C d) A:T and C:G e) C:G and A:C

88 Which of the following enzymes performs light dependent repair? a) DNA gyrase b) DNA polymerase c) DNA photolyase d) RNA polymerase e) DNA helicase

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