Chapter 12 Molecular Mechanisms of Mutation and DNA Repair

1. Chapter 12 Molecular Mechanisms of Mutation and DNA Repair

2. Mutations • A mutation is any heritable change in the genetic material • Mutations are classified in a variety of ways • Most mutations are spontaneous—they are random, unpredictable events • Each gene has a characteristic rate of spontaneous mutation, measured as the probability of a change in DNA sequence in the time span of a single generation

3. Table 12.1 Table 12.1: Major types of mutations and their distinguishing features

4. Mutations • Rates of mutation can be increased by treatment with a chemical mutagen or radiation, in which case the mutations are said to be induced • Mutations in cells that form gametes are germ-linemutations; all others are somatic mutations • Germ-line mutations are inherited; somatic mutations are not • A somatic mutation yields an organism that is genotypically a mixture (mosaic) of normal and mutant tissue

5. Mutations • Among the mutations that are most useful for genetic analysis are those whose effects can be turned on or off by the researcher • These are conditional mutations: they produce phenotypic changes under specific (permissive conditions) conditions but not others (restrictive conditions) • Temperature-sensitive mutations: conditional mutation whose expression depends on temperature

6. Figure 12.1: Siamese cat Courtesy of Jen Vertullo

7. Mutations • Mutations can also be classified according to their effects on gene function: • A loss-of-function mutation (a knockout or null) results in complete gene inactivation or in a completely nonfunctional gene product • A hypomorphic mutation reduces the level of expression of a gene or activity of a product • A hypermorphic mutation produces a greater-than-normal level of gene expression because it changes the regulation of the gene so that the gene product is overproduced • A gain-of-function mutation qualitatively alters the action of a gene. For example, a gain-of-function mutation may cause a gene to become active in a type of cell or tissue in which the gene is not normally active.

8. Figure 02A: An adult head in which both antennae form eye structures Reproduced from G. Halder, P. Callaerts, and W. J. Gehring, Science 267 (1995): 1788-1792. Reprinted with permission from AAAS. [http://www.sciencemag.org/].

9. Figure 02B: A wing with eye tissue growing out from it Reproduced from G. Halder, P. Callaerts, and W. J. Gehring, Science 267 (1995): 1788-1792. Reprinted with permission from AAAS. [http://www.sciencemag.org/].

10. Figure 02C: A single antenna in which most of the third segment consists of eye tissue Reproduced from G. Halder, P. Callaerts, and W. J. Gehring, Science 267 (1995): 1788-1792. Reprinted with permission from AAAS. [http://www.sciencemag.org/].

11. Figure 02D: Middle leg with an eye outgrowth at the base of the tibia Reproduced from G. Halder, P. Callaerts, and W. J. Gehring, Science 267 (1995): 1788-1792. Reprinted with permission from AAAS. [http://www.sciencemag.org/].

12. Mutations • Mutations result from changes in DNA • A base substitution replaces one nucleotide pair with another • Transition mutations replace one pyrimidine base with the other or one purine base with the other. There are four possible transition mutations

13. Mutations • Transversion mutations replace a pyrimidine with a purine or the other way around. There are eight possible transversion mutations • Spontaneous base substitutions are biased in favor of transitions • Among spontaneous base substitutions, the ratio of transitions to transversions is approximately 2:1

14. Mutations • Mutations in protein-coding regions can change an amino acid, truncate the protein, or shift the reading frame: • Missense or nonsynonymous substitutions result in one amino acid being replaced with another • Synonymous or silent substitutions in DNA do not change the amino acid sequence • Silent mutations are possible because the genetic code is redundant

15. Mutations • A nonsense mutation creates a new stop codon • Frameshift mutations shift the reading frame of the codons in the mRNA • Any addition or deletion that is not a multiple of three nucleotides will produce a frameshift

16. Sickle-cell anemia • The molecular basis of sickle-cell anemia is a mutant gene for b-globin • The sickle-cell mutation changes the sixth codon in the coding sequence from the normal GAG, which codes for glutamic acid, into the codon GUG, which codes for valine • Sickle-cell anemia is a severe genetic disease that often results in premature death • The disease is very common in regions where malaria is widespread because it confers resistance to malaria

17. Figure 12.3: Molecular basis of sickle-cell anemia

18. Trinucleotide repeats • Genetic studies of an X-linked form of mental retardation revealed a class of mutations called dynamic mutations because of the extraordinary genetic instability of the region of DNA involved • The molecular basis of genetic instability is a trinucleotide repeat expansion due to the process called replication slippage

19. Figure 12.6: Model of replication slippage

20. Fragile-X Syndrome • The X-linked condition, is associated with a class of X chromosomes that tends to fracture in cultured cells that are starved for DNA precursors • They are called fragile-X chromosomes, and the associated form of mental retardation is the fragile-X syndrome • The fragile-X syndrome affects about 1 in 2500 children • The molecular basis of the fragile-X chromosome has been traced to the expansion of a CGG trinucleotide repeat present at the site where the breakage takes place

21. Figure 12.4: Pedigree showing transmission of the fragile-X syndrome Adapted from C. D. Laird, Genetics 117 (1987): 587-599.

22. Fragile-X Syndrome • Normal X chromosomes have 6–54 tandem copies of CGG, whereas affected persons have 230–2300 or more copies • An excessive number of copies of the CGG repeat cause loss of function of a gene designated FMR1(fragile-site mental retardation-1) • Most fragile-X patients exhibit no FMR1 mRNA • The FMR1 gene is expressed primarily in the brain and testes

23. Figure 12.5: Dynamic mutation

24. Dynamic Mutations and Diseases • Other genetic diseases associated with dynamic mutation include: • The neurological disorders myotonic dystrophy (with an unstable repeat of CTG) • Kennedy disease (AGC) • Friedreich ataxia (AAG) • Spinocerebellar ataxia type 1 (AGC) • Huntington disease (AGC)

25. Transposable Elements • In a 1940s study of the genetics of kernel mottling in maize, Barbara McClintock discovered a genetic element that could move (transpose) within the genome and also caused modification in the expression of genes at or near its insertion site. • Since then, many transposable elements (TEs) have been discovered in prokaryotes and eukaryotes • They are grouped into “families” based on similarity in DNA sequence

26. Transposable Elements • The genomes of most organisms contain multiple copies of each of several distinct families of TEs • Once situated in the genome, TEs can persist for long periods and undergo multiple mutational changes • Approximately 50% of the human genome consists of TEs; most of them are evolutionary remnants no longer able to transpose

27. Transposable Elements • Some transposable elements transpose via a DNA intermediate others via an RNA intermediate • A target-site duplication is characteristic of most TEs insertions, and it results from asymmetrical cleavage of the target sequence • A large class of TEs called DNA transposons transpose via a cut-and-paste mechanism: the TE is cleaved from one position in the genome and the same molecule is inserted somewhere else

28. Figure 12.8: The sequence arrangement of a cut-and-paste transposable element and the changes that take place when it inserts into the genome

29. Transposable Elements • Each family of TEs has its own transposase—an enzyme that determines distance between the cuts made in the target DNA strands • Characteristic of DNA TEs is the presence of short terminal inverted repeats • Another large class of TEs possess terminal direct repeats, 200–500 bp in length, called long terminal repeats, or LTRs

30. Transposable Elements • TEs with long terminal repeats are called LTR retrotransposons because they transpose using an RNA transcript as an intermediate • Among the encoded proteins is an enzyme known as reverse transcriptase, which can “reverse- transcribe,” using the RNA transcript as a template for making a complementary DNA daughter strand • Some retrotransposable elements have no terminal repeats and are called non-LTR retrotransposons

31. Figure 12.10: Drosophila melanogaster

32. Transposable Elements • TEs can cause mutations by insertion or by recombination • In Drosophila, about half of all spontaneous mutations that have visible phenotypic effects result from insertions of TEs • Genetic aberrations can also be caused by recombination between different (nonallelic) copies of a TE

33. Figure 12.11: Recombination between transposable elements

34. Figure 12.12: Unequal crossing-over

35. Spontaneous Mutations • Mutations are statistically random events—there is no way of predicting when, or in which cell, a mutation will take place • The mutational process is also random in the sense that whether a particular mutation happens is unrelated to any adaptive advantage it may confer on the organism in its environment • A potentially favorable mutation does not arise because the organism has a need for it

36. Spontaneous Mutations • Several types of experiments showed that adaptive mutations take place spontaneously and were present at low frequency in the population even before it was exposed to the selective agent • One experiment utilized a technique developed by Joshua and Esther Lederberg called replica plating • Selective techniques merely select mutants that preexist in a population

37. Figure 12.13: Replica plating

38. Figure1 2.14: The ClB method for estimating the rate at which spontaneous recessive lethal mutations arise

39. Mutation Hot Spots • Mutations are nonrandom with respect to position in a gene or genome • Certain DNA sequences are called mutational hotspots because they are more likely to undergo mutation than others • For instance, sites of cytosine methylation are usually highly mutable

40. Figure 12.15: Spontaneous loss of the amino group

41. Mutagenes • Almost any kind of mutation that can be induced by a mutagen can also occur spontaneously, but mutagens bias the types of mutations that occur according to the type of damage to the DNA that they produce Table 12.3: Major agents of mutation and their mechanisms of action

42. Figure 12.16: Depurination

43. Figure 12.17: Deamination of adenine results in hypoxanthine

44. Figure 12.18: Mispairing mutagenesis by 5-bromouracil

45. Figure 12.19: Two pathways for mutagenesis by 5-bromouracil (Bu)

46. Figure 12.20: Chemical structures of two highly mutagenic alkylating agents

47. Figure 12.21: Mutagenesis of guanine by ethyl methanesulfonate (EMS)

48. Figure 12.22: Structural view of the formation of a thymine dimer

49. Figure 12.25: Mutation rates of five tandem repeats Data from Y. E. Dubrova, et al., Nature 380 (1996): 683-686.

50. DNA Repair Mechanisms • Many types of DNA damage can be repaired • Mismatch repairfixes incorrectly matched base pairs • The AP endonuclease system repairs nucleotide sites at which the base has been lost • Special enzymes repair damage caused to DNA by ultraviolet light • Excision repair works on a wide variety of damaged DNA • Postreplication repair skips over damaged bases