Announcements

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

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 pyrimidinereplaced 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 orrecombination. Other examples: Ethidium Bromide, DAPI.

5. Deamination is Caused by Nitrous Acid

(a) Causes: C -> U/T transition (and G -> A transition)(b) Causes: A -> G transition (and T -> C transition).

Deamination can be spontaneous as well.

6. Ultraviolet Radiation CauseThymine Dimers

260 nanometerwavelength

Disrupts synthesis;good for sterilizationof bacteria, bad for skin cancer.

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 childrennow- 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

PhotoreactivationEnzyme Cleavesbond between T’s

UV

Visible light

Base Excision Repair in Prokaryotes

DNA glycosylase

Apyrimidinic endonuclease

DNA Pol I, DNA ligase

Nucleotide Excision Repair:Prokaryotes and Eukaryotes

DNA Pol I

DNA ligase

uvr nuclease

Model for Eukaryotic Nucleotide Excision Repair: Xeroderma Pigmentosum

Mottled redness andpigmentation sign ofdamage due to UVexposure. Precursorto cancer. 4 years old.

Carefully protected fromsunlight. 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