1. Announcements http://cwx.prenhall.com/bookbind/pubbooks/klug3/
Self-grading problems = good practice for exam

2. Review of Last Lecture
1. Post-transcriptional gene regulation: Alternative splicing
2. Classification of mutations
3. Detection of mutations in humans
4. Different forms of mutations

3. Outline of Lecture 29 I. Origins of mutation
II. Mechanisms of DNA repair
III. Transposable elements

4. I. Different origins of mutation
1. Tautomeric Shifts: spontaneous
2. Base Analogues: chemical
3. Alkylating Agents: chemical
4. Intercalating Dyes: chemical
5. Deamination: chemical
6. UV Radiation and Thymine Dimers
7. High-Energy Radiation (X rays, gamma rays, cosmic rays)
environmental

5. Mispairing Due to Tautomeric Shifts

6. Formation of a TA to CG Transition During DNA Replication
Transition is a purine replaced by different purine or pyrimidine
replaced by different pyrimidine.

7. 2. Base Analogues: DNA can Incorporate 5-BU in place of Thymine
common
rare
Changes T-A pair > C-G pair. T > C, and A > G are both Transitions

8. 3. Alkylating Agents: Ethylmethane Sulfonate (EMS) Alkylates Guanine
Note: changes a G-C pair into an A-T pair
(G > A is a transition, C > T is a transition)
Another example: mustard gases first used in WWII.

9. 4. Intercalating Dyes Cause Frameshifts
Intercalate themselves into the DNA double helix, distorting it,
and causing insertion or deletion during DNA replication or
recombination. Other examples: Ethidium Bromide, DAPI.

10. 5. Deamination is Caused by Nitrous Acid
(a) Causes: C -> U/T transition (and G -> A transition)
Causes: A -> G transition (and T -> C transition).
Deamination can be spontaneous as well.

11. 6. Ultraviolet Radiation Cause
Thymine Dimers
Disrupts synthesis;
good for sterilization
of bacteria, bad for skin cancer.
260 nanometer
wavelength

12. 7. High-Energy (Ionizing) Radiation

13. Effects of Ionizing Radiation
Causes either point mutations or breaks in phosphodiester bonds of DNA backbone.
If both strands broken, there can sometimes be repair in mammals through the double-strand break repair (DSB) system.
Dividing cells are more susceptible to therapeutic X-rays than non-dividing cells (radiation therapy for cancer).

14. Example of ionizing radiation
nuclear reactor in Chernobyl, Soviet Union overheated, exploded, and ejected radioactive material into the environment - largest radiation accident in world.
Gamma rays emitted from radioactive elements are a source of ionizing radiation: 31 killed, acute radiation sickness, longterm effects ???
No increase so far in # of leukemias, but significant increase in # of childhood thyroid cancers
normal cases / million children
now- 100 cases / million children

15. Ionizing radiation transforms stable atoms into reactive free radicals and ions, which cause mutations in DNA

16. Radiation Doses in Perspective
Category Dose (mSv, millisievert)
Lethal full-body dose 3000
Detectable increase in cancer > 200
Chernobyl cleanup worker 250
Chernobyl nearby resident 50
Germany resident
Average yearly medical 0.39
Yearly background 2-3
Yearly smoking 2.8

17. Irradiation of Food (and Mail?)
Food/mail is exposed to X rays or electrons; doesn’t contact radioactive material or become radioactive itself.
Pro:
Prevents spoilage of food.
Reduces bacterial food-borne diseases.
Reduces reliance on chemical preservatives.
Con:
Produces chemical changes in food (so do other preservation methods); safe?
Selects for radiation-resistant bacteria?

18. II. Mechanisms of DNA Repair
Prokaryotic:
Photoactivation Repair
Base Excision Repair
Post-replication Repair and SOS Repair
Eukaryotic:
Nucleotide Excision Repair
Proofreading and Mismatch Repair
Double-Strand Break Repair

19. Photoactivation Repair in ProkaryotesUV
Photoreactivation
Enzyme Cleaves
bond between T’s
Visible light

20. Base Excision Repair in Prokaryotes
DNA glycosylase
Apyrimidinic endonuclease
DNA Pol I, DNA ligase

21. Nucleotide Excision Repair: Prokaryotes and Eukaryotes
uvr nuclease
DNA Pol I
DNA ligase

22. Model for Eukaryotic Nucleotide Excision Repair: Xeroderma Pigmentosum
Mottled redness and
pigmentation sign of
damage due to UV
exposure. Precursor
to cancer. 4 years old.
Carefully protected from
sunlight. 18 years old.
Works as a model.
7 Complementation Groups deficient in Excision Repair identified by Cell-cell Hybridization.

23. Proofreading and Mismatch Repair
Most DNA polymerases contain “proofreading” activity (3’ to 5’ exonuclease); increases fidelity of replication by 100X.
Remaining errors fixed by Mismatch Repair:
How does system recognize which strand is correct for use as template?
In bacteria, old strands become methylated, repair system recognizes unmethylated new strands. Similar system may work in eukaryotes.

24. Post-Replication Repair in Prokaryotes involves an Intentional Recombination

25. Double-Strand Break Repair in Eukaryotes
When both strands are broken and template can’t be used to repair the damage, DSB repair pathway reanneals two DNA segments.
Homologous recombination repair uses DNA from undamaged homologue (!)
Nonhomologous recombination repair also occurs.
Defects in this pathway associated with X-ray hypersensitivity and immune deficiency.

26. Site-directed mutagenesis
Using mutations to study gene function in the lab
Change 1 or more nucleotides in a gene to change a triplet codon and thus the amino acid sequence of the protein
Introduce mutated gene into animal
Determine effect on gene expression and protein function

28. III. Transposable Elements
Also called Transposons or “Jumping Genes”; can move within the genome.
Present in all organisms; well-studied in bacteria, maize, flies.
Discovered in Maize Ac-Ds system:

29. Corn Kernel Pigmentation Phenotype Caused by Ac-Ds Transposition: Colored Aleurone

30. Barbara McClintock, 1983 Nobel Prize in Medicine or Physiology

31. Mendel’s wrinkled Phenotype in Peas Also Caused by Transposon

32. P elements in Drosophila
P elements are transposons in flies.
The can be used experimentally create mutants, mark the positions of genes, or clone genes.
They can also be used to insert genes into the genome, creating a transgenic fly.

33. Transposons in Humans
Alu family of short interspersed elements (SINEs)
Moderately repetitive DNA
500,000 copies of bp repeats
Medical example: a transposon jumped into the gene on X chromosome responsible for hemophilia
Not present on either X chromosome of mother
Present on chromosome 22 of mother