1. Mutation and DNA Repair

2. Mutation Rates Vary Depending on Functional Constraints

3. Low Mutation Rates are Necessary for the Evolution of Complexity 1. Because most mutations are deleterious, there are limits to the number of mutations that an organism can afford to accumulate in its somatic body, e.g., a) given mean eukaryotic rates, genomes can accommodate 60,000 genes without intolerable mutational loads (Alberts et al.) b) a mutation rate 10 times higher would limit genome size to ca. 6000 genes 2. Both the germ line and the somatic body must be protected from mutational load (rare mutations become common because of large genomes and cell proliferation), e.g., a) germ line: (1) DNA repair (2) meiotic recombination in all eukaryotes (3) sequestering of germ line in metazoans (4) diplontic selection among cell lineages in meristems of plants b) somatic tissues...20% of deaths in western societies are due to cancer (uncontrolled cell proliferation) resulting largely to the accumulation of genetic damage in somatic tissues (1) DNA repair (2) immune systems 3. less efficient DNA repair and absence of meiosis may explain the limitation of prokaryotes to small genomes and unicellular forms before the origin of these processes in the protoeukaruote line. 4. spontaneous nucleotide changes are much higher than mutation rates would indicate, because of DNA repair mechanisms

4. Two strategies to study gene function Genotype to Phenotype - sequencing and searching for homologous sequences, then study their function Phenotype to Genotype - mutational screens and functional analysis

5. Kinds of Mutations Point Mutations –Same sense mutations –Missense Mutations –Nonsense Mutations –Transitions –Transversions Frame shift mutations Substitutions, Deletions and Additions

6. Chemistry of single nucleotide substitutions: a) transitions: a pyrimidine replaces a pyrimidine (C  T or T  C) or a purine replaces a purine (A  G or G  A) b) transversions: a pyrimidine replaces a purine or vice versa c) transitions are less severe mutations that transversions: (1) chemically, purines are more similar to one another than they are to pyrmidines, and vice versa (2) genetically, amino acid substitution is less likely with transitions because of the degeneracy of the genetic code (a) 3rd position transitions often code same amino acid i) UUU and UUG both code for leucine ii) GAA and GAG both code for glutamic acid (b) 3rd position transversion less often codes for same amino acid i) UUU and UUG code for phenylanaline and leucine

7. Mutagenesis Spontaneous Mutations –Replication Errors –Other Errors Chemical Mutagenesis Radiation-induced Mutations

8. Replication Errors

9. Replication Proofreading

10. Mutator Strains of E. coli error prone replication mutD codes for  subunit of DNA pol III:

11. DNA polymerase III holoenzyme with subunits (weight in daltons)  Step 1: previous nucleotide pair is tested for complementarity. If passed, elongation occurs. Step 2: If failed, the elongating strand is transferred to the exonuclease site to excise the mismatched nucleotide.

12. Experimental Demonstration of Proofreading artificial template double labeled probe last nucleotide is non- complementary and labeled non-complementary nucleotide excised, but no complementary nucleotides

13. Tautomerization of Bases

14. Thymine Tautomers: TA to TG binding mutation from T to A

15. Replication replication A T keto A T keto template daughter A T keto daughter template replication ATAT ATAT ATAT ATAT A T enol A T keto template daughter G T enol daughter template replication ATAT ATAT GCGC ATAT if unrepaired

16. Adenine Tautomers : AT to AC binding mutation from C to T

17. Replication replication A amino T A amino T template daughter A amino T daughter template replication ATAT ATAT ATAT ATAT A imino T A imino C template daughter A amino T daughter template replication ATAT GCGC ATAT ATAT if unrepaired

18. Cytosine Tautomers : C amino G C imino A binding mutation from C to T common results in CG pairing rare results in CA pairing AT substitution

19. Replication replication C amino G C amino G template daughter C amino G daughter template replication CGCG CGCG CGCG CGCG C imino G C imino A template daughter C amino G daughter template replication CGCG TATA CGCG CGCG if unrepaired

20. Guanine Tautomers : G keto C G enol T binding mutation from G to A common results in GC pairing rare results in GT pairing

21. Replication replication G keto C G keto C template daughter G keto C daughter template replication GCGC GCGC GCGC GCGC G enol C G enol T template daughter G keto C daughter template replication GCGC TATA GCGC GCGC if unrepaired

22. Frameshift Mutations insertion

23. Mechanism of Frameshift Mutation: “Slipping a cog” …a base fails to pair with its partner during replication

24. Spontaneous Mechanisms Outside of Replication

25. Spontaneous hydrolysis can result in deamination and depurination

26. Deamination replacement of an amino group by a carbonyl oxygen These nucleotide analogs have different pairing affinities, but analogs can be recognized and repaired 5-methyl C deamination results in T, which can’t be recognized as a mutation Replication produces a GC and an AT C’s are selected for methylation in certain CG sequences, which has led to the conversion of most CG’s to TG’s during evolution

27. Deamination of C and A illustrating different pairing behavior

28. Deamination and repair of C Deamination

29. Repair of a Deaminated Cytosine

30. Deamination of 5-methylcytosine

31. Triplet Repeats

32. Pathology results when repeats exceed a threshold number.

33. Amplification of copy number by unequal crossing-over Unequal crossing-over becomes more likely with increased copy number

34. Dynamic Mutations Unequal crossing-over becomes more likely with increased copy number and The severity of the pathology increases with copy number therefore... Both the probability of the pathology and its severity increase over generations after the number of repeats approaches the threshold

35. A number of conditions are based on this mechanism operating in different genes

36. The repeats can be located in different orientations with regard to the coding sequence upstream downstream within

37. The repeats can be located in different orientations with regard to the coding sequence...even within a single gene

38. Chemical Mutagenesis

40. EMS is an alkylating agent

41. Nucleoside analogs can exhibit variant pairing behavior keto (above); enol pairs to G instead of A

42. Acridine dyes intercalate DNA sequences Effect: stabilizes the looping that leads to deletions and insertions that cause frame shift mutations

43. Mechanism of Frameshift Mutation: “Slipping a cog” …a base fails to pair with its partner during replication

44. Major Repair Mechanisms Mismatch repair Excision repair Double strand breaks repaired mainly by end-joining Inducible & error-prone mechanisms

45. Excision Repair

46. Excision repair mechanism

47. More excision repair modalities

48. Repair of UV damage

49. Thymine dimers

50. Excision Repair of UV Induced Thymine Dimers

51. Mismatch Repair

52. To catch single-base errors that slip through proofreading during replication Happens right after replication Misses CC and small insertions and deletions mutH, mutL, mutS mutator strains are involved in mismatch repair Trick is distinguishing the new daughter strand

53. Missmatch Repair MutH How is the daughter strand recognized as the strand to correct?

54. Mismatch Repair GATC sequences methylated on the 6 position of the A base

55. Mismatch Repair endonuclease Activity …nicks DNA

56. and then methylation

57. Radiation Induced Mutagenesis UV induced Thymine dimers Gamma and X-ray double stranded breaks

58. Spectrum

59. X-rays induce mutations

60. Multiple mechanisms to repair UV damage

61. Photo-activated Repair System

62. problem: deletion of short nucleotide sequence Repairing Double- stranded Breaks often caused by radiation (high energy gamma or X-rays, directly or by creation of free radicals) repaired by: ◊ Homologous recombination ◊ Blunt-end repair (right)

63. Inducible Repair Backup systems activated only in emergencies Inducible Error prone

64. SOS

65. Undoing alkylation Note that the enzyme is expended! A tangible example of the importance of DNA repair

66. Photo-activated Repair System

67. Recombination Repair bulky mutations can leave gapsafter replication

68. p53 activation of DNA repair