Understanding Mutations and Their Classification

1. Mutations • Mutations are inheritable changes in the DNA • “Failure to faithfully store genetic information” • Changes can be to chromosomes or genes • Current focus: changes to DNA sequences. • This means an alteration in a basepair or • in the order of the basepairs.

2. Types of mutations-1 • Mutations can be classified in many, many ways • Some ways mutually exclusive, some not. • Spontaneous vs. Induced • Spontaneous happens naturally • Enzymatic errors, especially in copying • Various chemical reactions • Induced mutations: specifically caused, as by researcher • Treatment with various chemicals, radiation • Gametic (germ line) vs. somatic • Gametic mutations can be passed on to next generation • Somatic only affects individual (in metazoans)

3. Types of mutations-2 • Morphological • Change in physical structure, readily observed • Nutritional/biochemical • Mutated enzyme results in phenotypic change • Bacterial auxotrophs; sickle cell anemia • Behavioral mutations • Regulatory mutations • Affect control of gene expression rather than protein

4. Types of mutations-3 • Lethal mutations: not easily studied unless: • Conditional mutations: expressed depending on environmental conditions, especially temperature. • a way to study lethal mutations: permissive and restrictive temperatures; esp. useful with bacteria • temp sensitive mutations occur naturally, continued in offspring: Siamese cats, Himalayan rabbits www.tcainc.org/photos/ farpoint/saavik1.jpg

5. Types of mutations-4 Classification of mutations by FUNCTION • Loss of function: knockout or null. • Hypomorphic: lowered expression, “leaky” • Hypermorphic: greater activity or more visible trait • typically regulatory mutation, results in increased expression • Gain of function: e.g. new enzymatic activity • a factor in evolution; • Dominant negative: bad apple spoils the bunch e.g. bad protein in multicomponent enzyme

6. Detection of mutations • Bacteria and fungi • Prototrophs and auxotrophs: microbe no longer able to synthesize or breakdown particular nutrient. • Change in behavior, e.g. motility • Various methods in plants and animals • Humans (“not suitable experimental organisms”) • Reliance on pedigrees • Possible to determine sex linkage, dominance

7. Mutations are rare (but not equally so) • Mutation rate depends on species and on gene • Hot spot: a location in DNA where mutations occur significantly more often than the usual 1/ 106. • Monotonous run of single nucleotide or tandem repeats: GGGGGGGGG or ATGGATGGATGG • Methylated cytosines • methylation is added a CH3 group to something • Cytosines are methylated to help indicate which DNA strand is older (helps with DNA repair). • Problem occurs when a cytosine is chemically damaged by deamination. (more later)

8. Mutations • Our example: • information, 3 letters at a time, read consecutively • Point mutations: • Frameshift mutations: Insertion

9. more Mutations • Frameshift: deletion • Transposon mutagensis: transposons are segments of DNA that can jump into another spot in the DNA; they have information.

10. More types of mutations • Switch between A & G, or C & T: transition • Switch between purine and pyrimidine: transversion • Silent: 3rd position of codon usually means same amino acid, so change here has no effect. • Missense: typically a single nucleotide change, causes change in amino acid and noticeable effect. • Nonsense: change amino acid codon to STOP codon • Additions, deletions, and “stuttering” • Stuttering: repeated sequences sometimes copied incorrectly; enzyme gets confused?

11. Mutagens: that which causes mutations • Base analogs: e.g. 5-bromouracil. In equilibrium between keto and enol forms • In keto form, looks like T • In enol form looks like C • Used one way, but when copied, mispairing can occur. • Modifying agents: chemically change bases • HNO2 nitrous acid: deaminates (amino to keto) • See upcoming slide: deamination • Alkylating agents (ethylmethane sulfonate): add methyl or ethyl group to bases (-CH3 or CH2CH3), cause mispairing during synthesis

12. Loss of a purine, a natural process Can lead to an incorrect base being added; a mutation. saturn.roswellpark.org/.../ AP_site_generation.gif

13. Pyrimidines and deamination Deamination: Loss of an amine group, replacement w/ a keto group. Deamination of cytosine makes uracil; recognized as wrong and repaired. Deamination of 5-methyl cytosine produces thymine, which is normal; results in a transition mutation.

14. Frameshift mutations • Cause misalignment during DNA replication; caused byintercalating agents such as ethidium bromide or acridine orange http://www.photobiology.com/photoiupac2000/pierard/intintercal.jpg

15. Radiation • UV light at 265 nm • causes thymine dimers; covalent connections between adjacent thymines. Hurried repair makes mistakes. • Ionizing radiation • short wavelength, high energy radiation, e.g. x-rays, gamma radiation. • Causes ss, ds breaks in DNA. http://academic.brooklyn.cuny.edu/ biology/bio4fv/page/molecular% 20biology/mutation-prym-dimers.jpeg

16. Ionizing Radiation • Major damage is from free radicals • Most abundant substance in cell is water; radiation produces radicals that attack DNA, causing breaks. • The effects of radiation are a matter of considerable scientific and political controversy. • Effects of high levels of radiation are well understood, but effects of low levels are very difficult to study. • Brief soapbox: after Chernobyl tragedy, people vacated many square miles around damaged reactor. Now, endangered animals making a comeback despite radiation.

17. Repair of DNA damage • Despite the constant bombardment of DNA with radiation and chemicals, cells possess repair mechanisms. • Repair systems exist for • UV light damage • Chemical changes to bases • Loss of bases • Incorrect copying • Ss and ds breaks in DNA http://earthobservatory.nasa.gov/Library/UVB/Images/dna_mutation.gif

18. Repair of Thymine dimers Photoreactivation: Enzyme uses a photon of blue light to separate thymines from each other. (When using UV as a mutagen, put cells in dark afterwards!) (in E. coli) Excision Repair: DNA repair enzymes recognize a distorted DNA helix (such as caused by thymine dimers). The entire local section of DNA is removed and replaced. In all prokaryotes & eukaryotes. http://www-personal.ksu.edu/~bethmont/excisio3.gif

19. Repair of chemical changes • Deamination of cytosine • as shown previously, converts cytosine to uracil • the enzyme uracil glycosylase cuts off uracil, leaving deoxyribose as part of backbone, creating an “AP” site • AP = apurinic or apyrmidinic, meaning purine etc. NOT there. • AP repair, mechanism that specifically fixes such places.

20. Creation of an AP site saturn.roswellpark.org/.../ AP_site_generation.gif

21. AP repair- continued Activity of uracil glycosylase or spontaneous loss of base from DNA can create an AP site. An endonuclease cuts out the remaining sugar-phosphate and replaces it with a complete nucleotide.

22. Bulky excision repair • Like in repair of UV-induced damage, cells sense bulges, kinks, or similar damage to DNA • Chunk of DNA containing the damaged area is excised, replaced by DNA polymerase I enzyme (or equivalent) • 13 bases removed in bacteria • Eukaryotes (always more elaborate) take out 28 nucleotides • In humans, failure in this repair system causes disease xeroderma pigmentosum with increased risk of skin cancer.

23. Mismatch repair If Proofreading misses: Other enzymes recognize that the wrong base pair is in place, cuts out incorrect one and replaces it. Which one is incorrect? Presumably the newest one = the one on the DNA chain with the least amount of methyl cytosines. cmgm.stanford.edu/.../DNA%20Repair%20-%20Doug/

24. SOS Repair • Especially in bacteria, when damage to DNA is severe, an emergency system goes into effect where damage is repaired rapidly, but sloppily. Introduces many mutations, some possibly fatal, but DNA damage would surely be fatal otherwise.