Michael M. Cox Jennifer A.

Doudna Michael ODonnell

Molecular Biology
Principles and Practice

DNA Mutation and Repair

Copyright 2012 by W. H. Freeman and Company

DNA repair pathways

Direct reversal of DNA damage Excise defective elements and replace with normal nucleotides

Repair of CPD Damages

Photolyase reverses UV-induced DNA damage by photoreactivation

T-T dimers flip out of the DNA helix into the photolyase active site
MTHF

Base Excision Repair

DNA glycosylases excise a damaged base

DNA glycosylase pathway

Adapted from Schaerer, O. D., Angew. Chem. Int. Ed. Engl. 34 (2003): 29462974.

Bulky Lesion Repair

Nucleotide excision repair

1. damage recognition

2. incision (cut) in the damaged DNA strand on each side of the lesion 3. excision (removal) of the oligonucleotide created by the incisions
4. synthesis of new DNA to replace excised DNA 5. ligation of the nick
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General pathway of nucleotide-excision repair

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Mismatch Repair

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Methylation labeling

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A model of early steps of methyldirected mismatch repair

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Completing methyl-directed mismatch repair

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Michael M. Cox Jennifer A. Doudna Michael ODonnell

Molecular Biology
Principles and Practice

Nucleic Acid Technologies

Copyright 2012 by W. H. Freeman and Company

Method 1: DNA Sequencing: dNTP vs ddNTP

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Dideoxynucleotide DNA sequencing

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Method 2: Cloning
Plasmids - small circular bacterial DNA capable of replication - in addition to main chromosome Transformation - process that inserts synthetic plasmid into bacteria (make pores in bacteria cell membrane with chemicals or electroporation; plasmid sneaks in)

plasmid

plasmid

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Cutting DNA and vector, recombination and making more vector (cloning)

purify product with gel electrophoresis or chromatography

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Cutting DNA and vector, recombination and making more vector (cloning)

cut vector and DNA with same restriction endonuclease complimentarity

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Cutting DNA and vector, recombination and making more vector (cloning)

Make cells permeable make it so vector can leak in through the cells outer membranes

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Method 3: Polymerase chain reaction (PCR)

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Summary of what I learned in this lecture

DNA repair pathways Repair of CPD pathways by photoreactivation using photolyase Base Excision Repair: Use DNA glycosylases to excise a damaged base Bulky Lesion Repair/Nucleotide Excision Repair: will use an excinuclease Mismatch Repair: Methylation labeling Nucleic Acid technologies: 1. Dideoxynucleotide DNA sequencing 2. Cloning 3. PCR (polymerase chain reaction)
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Practice Problems
1. Which of these enzymes is not directly involved in methyl-directed mismatch repair in E. coli? A)DNA glycosylase 2. When a mismatch is introduced in a double-stranded DNA, the methyl-directed repair system: D)corrects the mismatch by changing the newly replicated strand 3. In base-excision repair, the first enzyme to act is: DNA glycosylase 4. The excinuclease is essential in: D)bulky lesion repair 5. Explain the role of DNA glycosylases in DNA repair. DNA glycosylases are enzymes that work in base excision repair. They cleave the N-glycosylic bond between a base and a sugar. This will lead to an AP site also known as an abasic DNA.
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Practice Problems Continued

6. Briefly explain the difference between base-excision repair and nucleotide-excision repair. Base excision repair involves an enzyme DNA glycosylase it cleaves the N-glycoslic bond which will cause only the base to be removed this forms an AP site also known as an abasic DNA. Nucleotide excision repair will use an excinucleoase that will cleave the phosphodiester bond this will cause the entire nucleotide to be repaired (sugar, phosphate, and base). 7. In the lab, recombinant plasmids/vectors are commonly introduces into bacterial cells by: E)transformation 8. The PCR reaction mixture does not include: C)DNA ligase

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Practice problems continued

9. Describe the steps and reaction components required in a PCR experiment. Illustrate the steps in just one round. In a PCR experiment you can amplify a segment of the DNA of interest. PCR involves heating the DNA (this will denature it) then allowing it to cool in the presence of primers and adding a polymerase. This cycle is then repeated 20-30 times. 10. Though not specifically mentioned in the lecture, can you speculate on why a heat-stable polymerase would simplify the PCR experiment? This would simplify the experiment because the heat-sensitive polymerase needs to be added in at a cooled temperature otherwise it will be denatured. If you had a heat stable polymerase then you wouldnt have to cool the reaction it would still be able to be run at high temp.
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