Lect. 23: Genetic Change

1. Lect. 23: Genetic Change ongoing question: whether mutation causes random variation leading to adaptation, or whether the environment induces heritable adaptations. Lamarckism is the doctrine of inheritance of acquired characteristics The random mutation doctrine says that sometimes chance changes happen to be adaptive, thus altering phenotype by changing a protein Chodavarapu et al (2008) Escherichia coli Dps interacts with DnaA protein to impede initiation: a model of adaptive mutation. MOLEC MICROBIOL 67: 1331 Godoy et al (2007) UmuD and RecA directly modulate the mutagenic potential of the Y family DNA polymerase DinB. MOLEC CELL 6: 1058

2. The fluctuation test: Luria and Delbrück (1943) • An E. coli population that started from small number of cells would show different patterns of T1 resistance depending on which model is correct. • The adaptive theory says that cells are induced to become resistant to T1 when it is added. Therefore, the proportion of resistant cells would be the same for all cultures with the same genetic background • The mutation theory says that random events confer resistance to T1, whether the phage is present or not. Cultures will therefore show different numbers of T1–resistant cells, depending on when the resistance mutation(s) occurred • Luria and Delbrück observed fluctuating numbers of resistant bacteria from E. coli cultures • Conclusion: the random mutation model is correct.

3. Fluctuation: representation of a dividing population of T1 phage-sensitive wild-type E. coli At generation 4, T1 phage is added. (a) If the adaptation theory is correct, cells mutate only when T1 phage is added, so the proportions of resistant cells in duplicate cultures are the same. (b) If the mutation theory is correct, cells mutate independently of when T1 phage is added, so the proportions of resistant cells in duplicate cultures are different. Left: If one cell mutates to become resistant to T1 phage infection at generation 3, then 2 of the 16 cells at generation 4 are resistant to T1. Right: If one cell mutates to become resistant at generation 1, then 8 of the 16 cells at generation 4 are resistant to T1.

4. Replica plating (Lederbergs, 1952): detecting lethal mutations Auxotrophs: Cells are first grown on supplemented medium, and then the colonies transferred to minimal medium, as well as to a control plate of supplemented medium. Colonies that grow on supplemented, but not minimal, media are selected for further study. Conditional Mutations • Mutations in some genes (e.g., DNA repair/replication and RNA polymerases) are usually lethal, so these genes are studied by isolating conditional mutations. • Heat sensitivity is a common conditional mutation, in which a normal protein is produced at permissive temperature, and a nonfunctional protein results at the nonpermissive temperature. Screening is generally done by replica plating and incubation at different temperatures.

5. Allelic series: structure/function Exposure to physical mutagens plays a role in genetic research, where they are used to increase mutation frequencies to provide mutant organisms for study.

6. Mutations Defined • A mutation is a change in a DNA base pair or a chromosome. • Somatic mutations affect only the individual in which they arise. • Germ-line mutations alter gametes, affecting the next generation. • two general categories: base-pair substitutions and deletions or insertions (indels) • Mutations are quantified in two different ways: • Mutation rate is the probability of a particular kind of mutation as a function of time (e.g., number per gene per generation). • Mutation frequency is the number of times a particular mutation occurs in proportion to the number of cells or individuals in a population (e.g., number per 100,000 organisms).

7. b: cell counts (no gametes)

8. Types of base-pair substitution mutations Broadest class (not including indels): • Transitions convert a purine-pyrimidine pair to the other purine-pyrimidine pair (e.g., AT to GC or TA to CG). More common. • Transversions convert a purine-pyrimidine pair to a pyrimidine-purine pair (e.g., AT to TA, or AT to CG). Many spontaneous errors are corrected by the cellular repair systems, and so do not become fixed in DNA.

9. Base-pair substitutions in ORFs are also defined by their effect on the protein sequence • Effects can vary from none to severe. • Missense mutations have a base-pair change resulting in a different mRNA codon, and therefore a different amino acid in the protein. • Nonsense mutations change a codon in the ORF to a stop (nonsense) codon, resulting in premature termination of translation, and a truncated (often nonfunctional) protein, depending on how far downstream. (ochre)

10. Phenotypic effects may not occur, depending on specific AA change • Neutral mutations change a codon in the ORF, but the resulting amino acid substitution produces no detectable change in the function of the protein • e.g., AAA to AGA substitutes arginine for lysine. The amino acids have similar properties (basic), so the protein’s function may not be altered. • Silent or synonymous mutations occur when the mutant codon encodes the same amino acid as the wild-type gene, so that no change occurs in the protein produced (e.g., AAA and AAG both encode lysine, so this transition would be silent).

11. Indels produce frameshifts: usually null alleles When the reading frame is shifted, incorrect amino acids are usually incorporated. • Frameshift mutations result from insertions or deletions when the number of affected base pairs is not divisible by three. • Frameshifts may bring stop codons into the reading frame, creating a shortened protein. • Frameshifts may also result in read-through of stop codons, resulting in a longer protein.

12. A nonsense mutation and its effect on translation

13. Mechanism of action of an intergenic nonsense-suppressor mutation that results from the mutation of a tRNA gene tRNAs Typical tRNA suppressor mutations are in redundant tRNA genes, so the wild-type tRNA activity is not lost. In this example, a tRNA.Tyr gene has mutated so that the tRNA’s anticodon is changed from 3’-AUG-5’ to 3’-AUC-5’, which can read a UAG nonsense codon, inserting tyrosine in the polypeptide chain at that codon.

16. Normal Watson–Crick and non–Watson–Crick base pairing in DNA Bases are normally in the keto form, but sometimes can undergo tautomeric shift to form the abnormal (enol) form. The enol form can hydrogen bond with an incorrect partner due to different spatial positioning of the atoms involved in H–bonding During DNA replication, the keto form of G could “wobble pair” with a normal T, producing a GT pair. GT pairs are targets for correction by proofreading during replication, and by other repair systems. Only mismatches uncorrected before the next round of replication lead to mutations.

17. Result of mismatch caused by “wobble” base pairing In the next round of replication, G is likely to be back in its keto form, and so both G and A are likely to pair normally, producing one progeny DNA with a GC pair, and another with an AT pair.

18. In its normal state, 5BU pairs with adenine (a). In its rare state (b), 5BU pairs with guanine

19. Mutagenic effects of the base analog 5-bromouracil (5BU) • The two possible mutation mechanisms. 5BU induces transition mutations when it incorporates into DNA in one state (indicated by white letters on magenta) and then shifts to its alternate state during the next round of DNA replication

20. Spontaneous generation of indel mutants by DNA looping-out errors during replication DNA loops out from the template strand, generally in a run of the same base. DNA Pol skips the looped-out bases, creating a deletion mutation. If Pol adds untemplated base(s), new looping occurs, resulting in mutation.

21. Additions and deletions can be induced during replication • Intercalating agents induce mutations by inserting between adjacent bases in dsDNA • Frameshift mutation by addition, when agent inserts itself into template strand. • Frameshift mutation by deletion, when agent inserts itself into newly synthesizing strand • Point deletions and insertions in ORFs result in frameshift mutations. These mutations can show reversion with a second treatment (if opposite sign mutation introduced).

22. E.g. Trinucleotide repeat diseases • Caused by “slippage” during DNA replication. Loop out structures may form while maintaining complementary base paring between the parent strand and daughter strand • Repeats in multiples of three  can give altered protein. • In Fragile X, the promoter region trinucleotide repeat (CGG)n premutation is expanded in females and silenced by small RNAs. • 1 in 1500 males; 1/2500 females • Huntington’s chorea disease caused by expansion that produces an altered form of the Htt protein, resulting in neuronal cell death in select areas of the brain. e.g.Myotonic dystrophy; spinobulbar muscular atrophy

23. Spontaneous chemical changes include depurination and deamination of particular bases • Depurination removes the purine (A or G) from DNA by breaking the bond with its deoxyribose in the backbone. • Depurination is common; thousands per day per cell • If not repaired before the next round of replication, DNA polymerase may stall or dissociate. • Deamination removes an amino group from a base • Uracil is an abnormal base in DNA, and it will usually be repaired. • If uracil is not replaced, it will pair with an A during replication, resulting in a CG-to-TA transition. • Both prokaryotic and eukaryotic DNA have small amounts of 5-methylcytosine (5mC) in place of the normal C. • Deamination of 5mC produces T. • T is a normal nucleotide in DNA, so it is not detected by repair mechanisms. • Deamination of 5mC results in CG-to-TA transitions.

24. 5-meC in lacI are hotspots for GC to AT transition • Locations of 5mC in the chromosome are often detected as mutational “hot spots” • Nonsense mutations shown; * are 5-MeC; open bars are potential nonsense not found. Of 50 independent mutations, 44 were at 5-MeC

25. Action of base-modifying agents (a) Nitrous acid modifies guanine, cytosine, and adenine. The cytosine and adenine modification results in mutations, but the guanine modification does not. (b) Hydroxyl-amine reacts only with cytosine. revertants do not occur with a second treatment. can be reverted by agents that do cause TA-to-CG transitions (e.g., 5BU and HNO2). Mutations induced by HNO2 can revert with a second treatment.

26. Alkylating agents produce transitions Usually alkylation occurs at the 6-oxygen of G, producing O6-alkylguanine. • Common mutagen for geneticists (very active) • Methyl or ethyl methane sulfonates (MMS, EMS) alkylates guanine and thymine, giving rise to GC AT and TA CG transitions, respectively • Can be reverted by alkylating agents or agents that do cause TA-to-CG transitions e.g., 5BU and HNO2

27. Three modifying mutagenic chemicals differentially affect bases lacI Amber = UAG

28. The Ames test for assaying the potential mutagenicity of chemicals • Simple and inexpensive screen for potential carcinogens. It assays the reversion rate of mutant strains of Salmonella typhimurium back to wild type • Histidine (his) auxotrophs are tested for reversion in the presence of the subject chemical by plating on media lacking the amino acid histidine. • Different his tester strains are available to test for base-substitution and frameshift mutations. • Liver enzymes (the S9 extract) are mixed with the test chemical to determine whether the liver’s detoxification pathways convert it to a mutagenic form. • More revertants in the region of the test chemical than in the untreated control indicate that it may be a mutagen, and further tests are indicated. • The Ames test is used routinely to screen industrial and agricultural chemicals, and shows a good, but not perfect, correlation between mutagens and carcinogens. • The test can be made quantitative to produce a dose-response curve, allowing comparison of relative mutagenicity of different chemicals. substitution strain frameshift strain

29. DNA Repair • Both prokaryotes and eukaryotes have enzyme-based DNA repair systems that prevent mutations and even death from DNA damage. • Repair systems are grouped by their repair mechanisms. Some directly reverse damage, while others excise the damaged area and then repair the gap. • Direct Reversal of DNA Damage • DNA polymerase proofreading corrects most of the incorrect nucleotide insertions that occur during DNA synthesis, which stalls until the wrong nucleotide is replaced with a correct one. • The role of 3’-to-5’ exonuclease activity is illustrated by mutator mutations in E. coli, which confer a much higher mutation rate on the cells that carry them. • The mutD gene, encoding the ε subunit of DNA polymerase III, is an example. Cells mutant in mutD are defective in proofreading. • Excision Repair • Dark repair (nucleotide excision repair) • Methyl-directed mismatch repair • Translesion DNA synthesis and SOS response

30. DNA glycosylase is a specific base excision repair mechanism • Base excision repair uses a repair glycosylase enzyme to recognize and remove damaged bases. • Bond between base and deoxyribose is cleaved by the glycosylase. • Other enzymes cleave sugar-phosphate from backbone, leaving a gap in the DNA. • Repair DNA polymerase and DNA ligase use the opposite strand as template to fill the gap.

31. Nucleotide excision repair (NER) of pyrimidine dimer and other damage-induced distortions of DNA • Aka “dark repair” (1964) • The proteins required are UvrA, UvrB, UvrC, and UvrD • A complex of two UvrA and one UvrB proteins (right X-tal) slides along the DNA. When it encounters a helix distortion, the UvrA subunits dissociate, and a UvrC binds the UvrB at the lesion. • When UvrBC forms, the UvrC cuts 4–5 nucleotides from the lesion on the 3’ side, and eight nucleotides away on the 5’ side. Then UvrB is released and UvrD binds the 5’ cut end. • UvrD is a helicase that unwinds the region between the cuts, releasing the short ssDNA, while DNA polymerase I fills the gap and DNA ligase seals the backbone. • In yeast and mammalian systems, about 12 genes encode proteins involved in excision repair.

32. Mechanism of mismatch repair • The mismatch correction enzyme recognizes which strand the base mismatch is on by reading the methylation state of a nearby GATC sequence. If the sequence is unmethylated, a segment of that DNA strand containing the mismatch is excised and new DNA is inserted. • Eukaryotes also have mismatch repair, but it is not clear how old and new DNA strands are identified.

33. Radiation (e.g., X-rays and UV) induces mutations Production of thymine dimers by ultraviolet light irradiation. The enzyme photolyase in prokaryotes plus 320 nm light can reverse reaction • X-rays are an example of ionizing radiation, which penetrates tissue and collides with molecules, knocking electrons out of orbits and creating ions (Muller, 1946 N.P.). • Ionizing radiation is the leading cause of human gross chromosomal mutations. • Ionizing radiation kills cells at high doses, and lower doses produce point mutations. • Ionizing radiation has a cumulative effect. A particular dose of radiation results in the same number of mutations whether it is received over a short or a long period of time. • UV is not energetic enough to induce ionization. • UV has lower-energy wavelengths than do X-rays, and so has limited penetrating power. • However, UV in the 254–260 nm range is strongly absorbed by purines and pyrimidines, forming abnormal chemical bonds.

34. Translesion DNA synthesis: Survival at the cost of incurring new mutations • Allows a cell to survive when specific base-pairing cannot occur • Bacteria use a system called SOS. In E. coli SOS is controlled by two genes, lexA and recA. Mutants in either of these genes have their SOS response permanently turned on. • When no DNA damage is present, LexA represses transcription of about 17 genes with products involved in various types of DNA repair. • Sufficient DNA damage activates the RecA protein, which stimulates LexA to autocleave, removing repression of the DNA repair genes. • After damage is repaired, RecA is inactivated, and newly synthesized LexA again represses the DNA repair genes. • The SOS system is a mutagenic bypass synthesis system. • DNA polymerase for translesion synthesis is made in SOS response. It replicates over and past the lesion. • Nucleotides are incorporated at the lesion that may not match wild type, leading to mutations. • This is better than the alternative, death due to incompletely replicated DNA.

35. Xeroderma pigmentosum disorder in humans occurs in homozygotes for a mutation in a repair gene. Other diseases also caused by DNA repair probs. • Affected individuals are photosensitive, and portions of skin exposed to light show intense pigmentation and warty growths that may become malignant. • Defects are in excision repair, and the inability to repair radiation damage to DNA often results in neoplastic malignancies.