GENE MUTATION AND DNA REPAIR

1. 1 GENE MUTATIONAND DNA REPAIR Chapter 16

2. 2 GENETIC MATERIAL DNA Primary function permanent storage of information Does not normally change Mutations do occur

3. 3 MUTATIONS Mutation Heritable change in the genetic material Permanent structural change of DNA Alteration can be passed on to daughter cells Mutations in reproductive cells can be passed to offspring

4. 4 MUTATIONS Mutations Provide allelic variation Ultimate source of genetic variation Foundation for evolutionary change Various phenotypic effects Neutral Harmful Beneficial

5. 5 MUTATIONS Mutations Most mutations are neutral More likely to be harmful than beneficial to the individual More likely to disrupt function than improve function

6. 6 MUTATIONS Mutations Many inherited diseases result from mutated genes Diseases such as various cancers can be caused by environmental agents known to cause DNA mutations �Mutagens�

7. 7 MODEL ORGANISMS Much of our understanding of mutations is a result of the study of model organisms e.g., Bacteria, yeast, Drosophila, etc. Amenable to analysis Short generation time, numerous offspring, etc. Often exposed to mutagenic environmental agents Effects of mutations are studied

8. 8 TYPES OF MUTATIONS Types of mutations Chromosome mutations Changes in chromosome structure Genome mutations Changes in chromosome number Single-gene mutations Relatively small changes in DNA structure Occur within a particular gene Focus of study in this chapter

9. 9 TYPES OF MUTATIONS Mutations involve the permanent alteration of a DNA sequence Alteration of base sequence Removal or addition of one or more nucleotides

10. 10 MUTATIONS Point mutations Change in a single base pair within the DNA Two main types of point mutations Base substitutions Transition Transversion Small deletions or insertions

11. 11 MUTATIONS Two types of base substitutions Transition Pyrimidine changed to another pyrimidine e.g., C ? T Purine changed to another purine e.g., A ? G Transversion Purines and pyrimidines are interchanged e.g., A ? C More rare than transitions

12. 12 EFFECTS OF MUTATIONS Mutations within the coding sequence of a gene can have various effects on the encoded polypeptide�s amino acid sequence Silent mutations Missense mutations Included neutral mutations Nonsense mutations Frameshift mutations

13. 13 EFFECTS OF MUTATIONS Silent mutations Amino acid sequence is not altered e.g., CCC ? CCG (pro ? pro) Genetic code is degenerate Alterations of the third base of a codon often do not alter the encoded amino acid Phenotype is not affected

14. 14 EFFECTS OF MUTATIONS Missense mutations Amino acid sequence is altered e.g., GAA ?GTA (glu ? val) Phenotype may be affected

15. 15 EFFECTS OF MUTATIONS Neutral mutations Type of missense mutation Amino acid sequence is altered e.g., CTT ?ATT (leu ? ile) e.g., GAA ?GAC (glu ? asp) No detectable effect on protein function Missense mutations substituting an amino acid with a similar chemistry to the original is likely to be neutral

16. 16 EFFECTS OF MUTATIONS Nonsense mutations Normal codon is changed into a stop codon e.g., AAA ? AAG (lys ? stop) Translation is prematurely terminated Truncated polypeptide is formed Protein function is generally affected

17. 17 EFFECTS OF MUTATIONS

18. 18 EFFECTS OF MUTATIONS

19. 19 EFFECTS OF MUTATIONS Mutations occasionally produce a polypeptide with an enhanced ability to function Relatively rare May result in an organism with a greater likelihood to survive and reproduce Natural selection may increase the frequency of this mutation in the population

20. 20 MUTATION TYPES Genetic terms to describe mutations Wild-type Relatively common genotype Generally the most common allele Variant Mutant allele altering an organism�s phenotype Forward mutation Changes wild-type allele into something else Reverse mutation �Reversion� Restores wild-type allele

21. 21 MUTATION TYPES Genetic terms to describe mutations Deleterious mutation Decreases an organism�s chance of survival Lethal mutation Results in the death of an organism Extreme example of a deleterious mutation Conditional mutants Affect the phenotype only under a defined set of conditions e.g., Temperature-sensitive (ts) mutants

22. 22 MUTATION TYPES Genetic terms to describe mutations Suppressor mutation Second mutation that restores the wild-type phenotype Intragenic suppressor Secondary mutation in the same gene as the first mutation Differs from a reversion Second mutation is at a different site than the first Intergenic suppressor Secondary mutation in a different gene than the first mutation

23. 23 MUTATION TYPES Two general types of intergenic suppressors Those involving an ability to defy the genetic code Those involving a mutant structural gene

24. 24 MUTATION TYPES Intergenic suppressor mutations involving an ability to defy the genetic code e.g., tRNA mutations Altered anticodon region e.g., Recognize a stop codon May suppress a nonsense mutation in a gene May also suppress stop codons in normal genes

25. 25 MUTATION TYPES Intergenic suppressors involving a mutant structural gene Usually involve altered expression of one gene that compensates for a loss-of-function mutation affecting another gene Second gene may take over the functional role of the first May involve proteins participating in a common cellular function Sometimes involve mutations in genetic regulatory proteins e.g., Transcription factors activating other genes that can compensate for the mutation in the first gene

26. 26 MUTATION TYPES Mutations occurring outside of coding sequences can influence gene expression Mutations may alter the core promoter sequence Up promoter mutations Mutant promoter becomes more like the consensus sequence Rate of transcription may be increased Down promoter mutations Mutant promoter becomes less like the consensus sequence Affinity for regulatory factors is decreased Rate of transcription may be decreased

27. 27 MUTATION TYPES Mutations occurring outside of coding sequences can influence gene expression Mutations may alter other regulatory sequences lacOC mutations prevent binding of the lac repressor Lac operon is constituently expressed, even in the absence of lactose Such expression is wasteful Such mutants are at a selective disadvantage

28. 28 MUTATION TYPES Mutations occurring outside of coding sequences can influence gene expression Mutations may alter splice junctions Altered order and/or number of exons in the mRNA

29. 29 MUTATION TYPES Mutations occurring outside of coding sequences can influence gene expression Mutations may affect an untranslated region of mRNA 5�- or 3�-UTR May affect mRNA stability May affect the ability of the mRNA to be translated

30. 30 MUTATION TYPES

31. 31 TRINUCLEOTIDE REPEATS DNA trinucleotide repeats Three nucleotide sequences repeated in tandem e.g., �CAGCAGCAGCAGCAGCAG� Generally transmitted normally from parent to offspring without mutation

32. 32 TRINUCLEOTIDE REPEATS Trinucleotide repeat expansion (TNRE) Number of repeats can readily increase from one generation to the next Cause of several human genetic diseases Length of a repeat has increased above a certain critical size Becomes prone to frequent expansion

33. 33 TRINUCLEOTIDE REPEATS TNRE disorders Fragile X syndrome (FRAXA) FRAXE mental retardation Myotonic muscular dystrophy (DM) Spinal and bulbar muscular atrophy (SBMA) Huntington disease (HD) Spinocerebellar ataxia (SCA1)

34. 34 TRINUCLEOTIDE REPEATS TNRE disorders

35. 35 TRINUCLEOTIDE REPEATS TNRE disorders Expansion may be within a coding sequence of a gene Most expansions are of a CAG repeat Encoded proteins possess long tracts of glutamine CAG encodes a glutamine codon Presence of glutamine tracts causes aggregation of the proteins Aggregation is correlated with the progression of the disease

36. 36 TRINUCLEOTIDE REPEATS TNRE disorders Expansion may be in a noncoding region of a gene Two fragile X syndromes Repeat produces CpG islands that become methylated Methylation can lead to chromosome compaction Can silence gene transcription Myotonic muscular dystrophy Expansions may cause abnormal changes in RNA structure

37. 37 TRINUCLEOTIDE REPEATS TNRE disorders Severity of the disease tends to worsen in future generations �Anticipation� Severity of the disease depends on the parent from whom it was inherited e.g., In Huntingdon disease, TNRE likely to occur if mutation gene is inherited from the father e.g., In myotonic muscular dystrophy, TNRE likely to occur if mutation gene is inherited from the mother

38. 38 TRINUCLEOTIDE REPEATS TNRE disorders

39. 39 TRINUCLEOTIDE REPEATS TNRE disorders Cause of TNRE is not well understood Trinucleotide repeat may produce alterations in DNA structure e.g., Stem-loop formation May lead to errors in DNA replication TNRE within certain genes alters gene expression Disease symptoms are produced

40. 40 CHROMOSOME STRUCTURE Altered chromosome structure can alter gene expression Inversions and translocations commonly have no obvious phenotypic effects Phenotypic effects sometimes occur �Position effect�

41. 41 CHROMOSOME STRUCTURE Altered chromosome structure can alter gene expression and phenotype Breakpoint may occur within a gene Expression of the gene is altered Breakpoint may occur near a gene Expression is altered when moved to a new location May be moved next to regulatory elements influencing the expression of the relocated gene i.e., Silencers or enhancers May reposition a gene from a euchromatic region to a highly condensed (heterochromatic) region Expression may be turned off

42. 42 CHROMOSOME STRUCTURE Altered chromosome structure can alter gene expression and phenotype An eye color gene relocated to a heterochromatic region can display altered expression Gene is sometimes inactivated Variegated phenotype results

43. 43 SOMATIC VS. GERM-LINE The timing of mutations in multicellular organisms plays an important role Mutations may occur in gametes or a fertilized egg Mutations may occur later in life Embryonic or adult stages Timing can affect The severity of the genetic effect The ability to be passed from parent to offspring

44. 44 SOMATIC VS. GERM-LINE Animals possess germ-line and somatic cells Germ-line cells Cells giving rise to gametes Somatic cells All cells of the body excluding the germ-line cells e.g., Muscle cells, nerve cells, etc.

45. 45 SOMATIC VS. GERM-LINE Germ-line cells Germ-line mutations can occur in gametes Germ-line mutations can occur in a precursor cell that produces gametes All cells in the resulting offspring will contain the mutation

46. 46 SOMATIC VS. GERM-LINE Somatic cells Somatic mutations in embryonic cells can result in patches of tissues containing the mutation Size of the patch depends on the timing of the mutation Individual is a genetic mosaic

47. 47 CAUSES OF MUTATIONS Two causes of mutations Spontaneous mutations Result from abnormalities in biological processes Underlying cause lies within the cell Induced mutations Caused by environmental agents Cause originates outside of the cell

48. 48 CAUSES OF MUTATIONS Causes of spontaneous mutations Abnormalities in crossing over Aberrant segregation of chromosomes during meiosis Mistakes by DNA polymerase during replication Alteration of DNA by chemical products of normal metabolic processes Integration of transposable elements Spontaneous changes in nucleotide structure

49. 49 CAUSES OF MUTATIONS Induced mutations are caused by mutagens Chemical substances or physical agents originating outside of the cell Enter the cell and then alter the DNA structure

50. 50 CAUSES OF MUTATIONS

51. 51 CAUSES OF MUTATIONS Spontaneous mutations are random events Not purposeful Mutations occur as a matter of chance Some individuals possess beneficial mutations Better adapted to their environment Increased chance of surviving and reproducing Natural selection results in differential reproductive success The frequency of such alleles increases in the population

52. 52 CAUSES OF MUTATIONS Salvador Luria and Max Delbruck T1 is a bacteriophage able to infect E coli A small percentage of bacteria are resistant to T1 infection Heritable trait tonr (T1 resistance) Is this resistance due to spontaneous mutations or due to a physiological adaptation?

53. 53 CAUSES OF MUTATIONS Salvador Luria and Max Delbruck The question Is T1 resistance due to spontaneous mutations or due to a physiological adaptation?

54. 54 CAUSES OF MUTATIONS Salvador Luria and Max Delbruck Two competing theories Adaptation theory Rate of adaptation should be relatively constant Depends on exposure to bacteriophage tonr cells should be a relatively constant proportion of the total bacterial population Spontaneous mutation theory Number of tonr cells is dependent on timing of mutation tonr mutation occurring early in proliferation ? many tonr mutants found tonr mutation occurring late in proliferation ? fewer tonr mutants found Predicts greater variation in the number of tonr cells present in different populations

55. 55 CAUSES OF MUTATIONS Salvador Luria and Max Delbruck The experiment �Fluctuation test� Grew T1-susceptible bacteria in a flask and in several individual tubes Plated onto media with T1 phage Counted number of tonr colonies

56. 56 CAUSES OF MUTATIONS Salvador Luria and Max Delbruck The results Even distribution of tonr colonies from large flask Great fluctuation in number of tonr colonies from small tubes

57. 57 CAUSES OF MUTATIONS Salvador Luria and Max Delbruck The conclusion Results are consistent with the spontaneous mutation theory Timing of the mutation during the growth of a culture greatly affects the number of mutant cells

58. 58 CAUSES OF MUTATIONS Joshua and Ester Lederberg (1950s) Interested in the relationship between mutation and the environmental conditions shat select for mutations Scientists were unsure of the relationship Two competing hypotheses Directed mutation hypothesis Some scientists still believed that selective conditions could promote specific mutations Random mutation theory Mutations occur at random Environmental factors affecting survival select for those possessing beneficial mutations

59. 59 CAUSES OF MUTATIONS Joshua and Ester Lederberg (1950s) Plated large number of bacteria onto a master plate Contained no selective agent Transferred colonies to secondary plates containing selective agent (T1 phage) �Replica plating� Only mutant cells would grow

60. 60 CAUSES OF MUTATIONS Joshua and Ester Lederberg (1950s) Mutants occupied the same locations on all secondary plates Indicated that mutations occurred randomly in colonies growing on the nonselective master plate Ransom mutation theory is supported

61. 61 CAUSES OF MUTATIONS Mutation rate Likelihood that a gene will be altered by a new mutation Expressed as the number of new mutations in a given gene per generation Generally 1/100,000 � 1/billion 10-5 � 10-9

62. 62 CAUSES OF MUTATIONS Mutation rate Mutation rate is not a constant number Can be increased by environmental mutagens Induced mutations can increase beyond frequency of spontaneous mutations Mutation rates vary extensively between species Even vary between strains of the same species

63. 63 CAUSES OF MUTATIONS Mutation rate Some genes mutate at a much higher rate than other genes Some genes are longer than others Some locations are more susceptible to mutation Even single genes possess mutation hot spots More likely to mutate than other regions

64. 64 CAUSES OF MUTATIONS Mutation frequency Number of mutant alleles of a given gene divided by the number of alleles within a population Timing of mutations influences mutation frequency Timing does not influence mutation rate Mutation frequency depends both on mutation rate and timing of mutations Natural selection and genetic drift can further increase mutation frequencies

65. 65 CAUSES OF MUTATIONS Spontaneous mutations: Depurination Most common type of naturally occurring chemical change Reaction with water removes a purine (A or G) from the DNA �Apurinic site�

66. 66 CAUSES OF MUTATIONS Spontaneous mutations: Depurination ~10,000 purines lost per 20 hours at 37oC in a typical mammalian cell Rate of loss increased by agents causing certain base modification e.g., Attachment of alkyl (methyl, ethyl, etc.) groups Generally recognized by DNA repair enzymes Mutation may result if repair system fails

67. 67 CAUSES OF MUTATIONS Spontaneous mutations: Deamination of cytosines Other bases are not readily deaminated Removal of an amino group from the cytosine base Uracil is produced DNA repair enzymes generally remove this base Uracil is recognized as an inappropriate base Mutation may result if repair system fails Uracil hydrogen bonds with A, not G

68. 68 CAUSES OF MUTATIONS Spontaneous mutations: Deamination of cytosines Methylation of cytosine occurs in many eukaryotic species as well as prokaryotes Removal of an amino group from the 5-methyl cytosine produces thymine DNA repair enzymes cannot determine which is the incorrect base Hot spots for mutations are produced

69. 69 CAUSES OF MUTATIONS Spontaneous mutations: Tautomeric shifts Common, stable form of T and G is the keto form Interconvert to an enol form at a low rate Common, stable form of A and C is the amino form Interconvert to an imino form at a low rate

70. 70 CAUSES OF MUTATIONS Spontaneous mutations: Tautomeric shifts Enol and imino forms do not conform to normal base-pairing rules AC and GT base pairs are formed

71. 71 CAUSES OF MUTATIONS Spontaneous mutations: Tautomeric shifts Tautomeric shifts immediately prior to DNA replication can cause mutations Resulting mismatch could be repaired Mutation may result if repair system fails

72. 72 CAUSES OF MUTATIONS Hermann Muller (1927) Showed that X rays can cause induced mutations Reasoned that a mutagenic agent might form defective alleles Experimental approach focused on formation and detection of X-linked genes in Drosophila melanogaster

73. 73 CAUSES OF MUTATIONS Hermann Muller (1927) The hypothesis Exposure to X rays will increase the rate of mutation

74. 74 CAUSES OF MUTATIONS Hermann Muller (1927) The materials Drosophila melanogaster strain with three genetic alterations of the X chromosome �ClB chromosome� C: Large inversion preventing productive crossing over l: Lethal recessive X-linked gene B: Bar eye allele

75. 75 CAUSES OF MUTATIONS Hermann Muller (1927) The design Females with one ClB chromosome and one normal chromosome Cannot undergo crossovers in the �C� region Can only produce sons possessing the normal X chromosome Lethal mutations in the �normal� chromosome will prevent them from producing any sons

76. 76 CAUSES OF MUTATIONS Hermann Muller (1927) The experiment Exposed wild-type males to X rays May mutate the X chromosome in sperm Mated mutagenized males to females with the ClB chromosome Daughters with bar eyes were saved Mated to wild-type males Genders of offspring determined

77. 77 CAUSES OF MUTATIONS Hermann Muller (1927) The data

78. 78 CAUSES OF MUTATIONS Hermann Muller (1927) Interpreting the data Very few lethal mutations occurred in the untreated control Approximately 1 in 1,000 Many more mutations occurred in the X ray-treated flies Nearly 100 times more X rays greatly increase the rate of X-linked recessive lethal mutations

79. 79 CAUSES OF MUTATIONS The public is concerned about mutagens for two important reasons Mutagenic agents are often involved in the development of human cancers Avoiding mutations that may have harmful effects on future offspring is desirable

80. 80 CAUSES OF MUTATIONS An enormous array of agents can act as mutagens Chemical agents and physical agents

81. 81 CAUSES OF MUTATIONS Certain non-mutagenic chemicals can be altered to a mutagenically active form after ingestion Cellular enzymes such as oxidases can activate some mutagens Certain foods contain chemicals acting as antioxidants Antioxidants may be able to counteract the effects of mutagens and lower cancer rates

82. 82 CAUSES OF MUTATIONS Mutagens alter DNA structure in various ways Nitrous acid (HNO3) replaces amino groups with keto groups -NH2 ? =O Can change cytosine to uracil Pairs with A, not G Can change adenine to hypoxanthine Pairs with C, not T

83. 83 CAUSES OF MUTATIONS Mutagens alter DNA structure in various ways Alkylating agents covalently attach methyl or ethyl groups to bases e.g., Nitrogen mustards, ethyl methanesulfonate (EMS) Appropriate base pairing is disrupted

84. 84 CAUSES OF MUTATIONS Mutagens alter DNA structure in various ways Some mutagens directly interfere with the DNA replication process e.g., Acridine dyes such as proflavin Flat, planar structures interchelate into the double helix Sandwich between adjacent base pairs Helical structure is distorted Single-nucleotide additions and deletions can result

85. 85 CAUSES OF MUTATIONS Mutagens alter DNA structure in various ways Some mutagens are base analogs e.g., 2-aminopurine e.g., 5-bromouracil (5BU) Become incorporated into daughter strands during DNA replication

86. 86 CAUSES OF MUTATIONS Mutagens alter DNA structure in various ways Some mutagens are base analogs 5-bromouracil (5BU) is a thymine analog Incorporated in place of thymine 5BU can base-pair with adenine Can tautomerize and base-pair with guanine at a relatively high rate AT ? A5BU ? G5BU ? GC Transition mutations occur

87. 87 CAUSES OF MUTATIONS Mutagens alter DNA structure in various ways DNA molecules are sensitive to physical agents such as radiation e.g., Ionizing radiation such as X rays and gamma rays Short wavelength and high energy Can penetrate deeply into biological materials Creates �free radicals� Chemically reactive molecules Free radicals alter DNA structure in a variety of ways Deletions, single nicks, cross-linking, chromosomal breaks

88. 88 CAUSES OF MUTATIONS Mutagens alter DNA structure in various ways DNA molecules are sensitive to physical agents such as radiation e.g., Nonionizing radiation such as UV light Contains less energy Penetrates only the surface of material such as the skin Causes the formation of thymine dimers May be repaired through one of numerous repair systems May cause a mutation when that DNA strand is replicated

89. 89 CAUSES OF MUTATIONS Many different kinds of testes can determine if an agent is mutagenic Ames test is commonly used Developed by Bruce Ames Uses his- strains of Salmonella typhimurium Mutation is due to a point mutation rendering an enzyme inactive Reversions can restore his+ phenotype Ames test monitors rate of reversion mutations

90. 90 CAUSES OF MUTATIONS Ames test Suspected mutagen is mixed with rat liver extract and his- Salmonella typhimurium Rat liver extract provides cellular enzymes that may be required to activate a mutagen Bacteria are plated on minimal media his+ revertants can be detected Mutation frequency calculated Compared to control

91. 91 DNA REPAIR Most mutations are deleterious DNA repair systems are vital to the survival Bacteria possess several different DNA repair systems Absence of a single system greatly increases mutation rate �Mutator strains� Humans defective in a single DNA repair system may manifest various disease symptoms e.g., Higher risk of skin cancer

92. 92 DNA REPAIR Living cells contain several DNA repair systems Able to fix different types of DNA alterations