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DNA: The Genetic MaterialChapter 14
Frederick Griffith – 1928
• Studied Streptococcus pneumoniae, a pathogenic bacterium causing pneumonia
• 2 strains of Streptococcus
– S strain is virulent
– R strain is nonvirulent
• Griffith infected mice with these strains hoping to understand the difference between the strains
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• Griffith’s results
– Live S strain cells killed the mice
– Live R strain cells did not kill the mice
– Heat-killed S strain cells did not kill the mice
– Heat-killed S strain + live R strain cells killed the mice
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• Transformation
– Information specifying virulence passed from the dead S strain cells into the live R strain cells
• Our modern interpretation is that genetic material was actually transferred between the cells
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Avery, MacLeod, & McCarty – 1944
• Repeated Griffith’s experiment using purified cell extracts
• Removal of all protein from the transforming material did not destroy its ability to transform R strain cells
• DNA-digesting enzymes destroyed all transforming ability
• Supported DNA as the genetic material
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Hershey & Chase –1952
• Investigated bacteriophages
– Viruses that infect bacteria
• Bacteriophage was composed of only DNA and protein
• Wanted to determine which of these molecules is the genetic material that is injected into the bacteria
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• Bacteriophage DNA was labeled with radioactive phosphorus (32P)
• Bacteriophage protein was labeled with radioactive sulfur (35S)
• Radioactive molecules were tracked • Only the bacteriophage DNA (as indicated
by the 32P) entered the bacteria and was used to produce more bacteriophage
• Conclusion: DNA is the genetic material
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DNA Structure
• DNA is a nucleic acid• Composed of nucleotides
– 5-carbon sugar called deoxyribose
– Phosphate group (PO4)
• Attached to 5′ carbon of sugar– Nitrogenous base
• Adenine, thymine, cytosine, guanine– Free hydroxyl group (—OH)
3. When the polymerase III on the lagging strand hits the previously synthesized fragment, it releases the β clamp and the template strand. DNA polymerase I attaches to remove the primer.
β clampreleases
Laggingstrandreleases
DNA polymerase III
DNA polymerase I
5´3´
5´3´
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Clamp loader
4. The clamp loader attaches the β clamp and transfers this to polymerase III, creating a new loop in the lagging-strand template. DNA ligase joins the fragments after DNA polymerase I removes the primers.
DNA ligasepatches “nick”
DNA polymerase Idetaches afterremoving RNA primer
5´3´
5´3´
5´3´
New bases
5. After the β clamp is loaded, the DNA polymerase III on the lagging strand adds bases to the next Okazaki fragment.
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Eukaryotic Replication
• Complicated by
– Larger amount of DNA in multiple chromosomes
– Linear structure
• Basic enzymology is similar
– Requires new enzymatic activity for dealing with ends only
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• Multiple replicons – multiple origins of replications for each chromosome– Not sequence specific; can be adjusted
• Initiation phase of replication requires more factors to assemble both helicase and primase complexes onto the template, then load the polymerase with its sliding clamp unit– Primase includes both DNA and RNA polymerase– Main replication polymerase is a complex of DNA
polymerase epsilon (pol ε) and DNA polymerase delta (pol δ)
Telomeres
• Specialized structures found on the ends of eukaryotic chromosomes
• Protect ends of chromosomes from nucleases and maintain the integrity of linear chromosomes
• Gradual shortening of chromosomes with each round of cell division– Unable to replicate last section of lagging
strand49
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• Telomeres composed of short repeated sequences of DNA
• Telomerase – enzyme makes telomere of lagging strand using and internal RNA template (not the DNA itself)– Leading strand can be replicated to the end
• Telomerase developmentally regulated– Relationship between senescence and telomere length
• Cancer cells generally show activation of telomerase
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DNA Repair
• Errors due to replication– DNA polymerases have proofreading ability
• Mutagens – any agent that increases the number of mutations above background level– Radiation and chemicals
• Importance of DNA repair is indicated by the multiplicity of repair systems that have been discovered
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DNA Repair
• Falls into 2 general categories1.Specific repair
– Targets a single kind of lesion in DNA and repairs only that damage
2.Nonspecific– Use a single mechanism to repair multiple
kinds of lesions in DNA
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Photorepair
• Specific repair mechanism• For one particular form of damage caused
by UV light• Thymine dimers
– Covalent link of adjacent thymine bases in DNA
• Photolyase– Absorbs light in visible range– Uses this energy to cleave thymine dimer
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Excision repair
• Nonspecific repair
• Damaged region is removed and replaced by DNA synthesis
• 3 steps1. Recognition of damage
2. Removal of the damaged region
3. Resynthesis using the information on the undamaged strand as a template