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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

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

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

Mispairing Due to Tautomeric Shifts

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

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

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.

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.

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.

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

7. High-Energy (Ionizing) Radiation

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).

Example of ionizing radiation 1986 - 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, 200 + acute radiation sickness, longterm effects ??? No increase so far in # of leukemias, but significant increase in # of childhood thyroid cancers normal - 0.5-3 cases / million children now- 100 cases / million children

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

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 0.4-0.9 Average yearly medical 0.39 Yearly background 2-3 Yearly smoking 2.8

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?

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

Photoactivation Repair in Prokaryotes UV Photoreactivation Enzyme Cleaves bond between T’s Visible light

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

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

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.

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.

Post-Replication Repair in Prokaryotes involves an Intentional Recombination

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.

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

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:

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

Barbara McClintock, 1983 Nobel Prize in Medicine or Physiology

Mendel’s wrinkled Phenotype in Peas Also Caused by Transposon

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.

Transposons in Humans Alu family of short interspersed elements (SINEs) Moderately repetitive DNA 500,000 copies of 200-300 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