Chapter 12 - DNA Structure & Replication
History of DNA
1869 Miescher
Analyzed the contents of the nucleus & discovered a substance (containing nitrogen &
phosphorus) which he called nuclein
1909 Garrod
Linked protein deficiencies to unusual phenotypes.
Could proteins be the genetic (hereditary) material?
1929 Griffith
Experimented with two phenotypically different bateria: R (rough coated) which
caused no disease & S (smooth coated) which would cause pneumonia
Mice injected with the R bacteria did not dies of pneumonia
Mice injected with the S bacteria died of pneumonia
When S bacteria were heat treated (to kill them), they no longer caused pneumonia
Makes sense, but here’s the kicker!
Chapter 12 - DNA Structure & Replication
Chapter 12 - DNA Structure & Replication
Chapter 12 - DNA Structure & Replication
Chapter 12 - DNA Structure & Replication
1929 Griffith (continued)
When heat treated S bacteria was co-injected with live R bacteria, the mice DIED of
pneumonia & LIVE S bacteria was found in the dead mice! What HAPPPENED?
Chapter 12 - DNA Structure & Replication
Chapter 12 - DNA Structure & Replication
1930s Avery, MacLeod, McCarty
Something in the heat-treated S bacteria transformed the R bacteria into live S bacteria
Mice were injected with a mixture of heat-treated S bacteria, live R bacteria & either:
1. A protease which destroys proteins
2. A DNAse which destroys DNA
Mice given the protease mixture died of pneumonia & live S were recovered
Mice given the DNAse mixture lived
Conclusion: S type DNA is capable of transforming (changing) live R into S type
Confirmation: They isolated DNA from the heat treated S bacteria & co-injected it with
live R into mice = mice died & contained living S bacteria
Chapter 12 - DNA Structure & Replication
1950 Hershey & Chase
Confirmed that DNA & not protein is the genetic material
Used viruses because:
Virus structure is simple: nucleic acid core surrounded by a protein coat
Once a virus infects a cell it causes the host cell to produce more viruses (this is the
phenotypic change that will indicate a transformation of the host cell’s genotype)
What part of the virus gets into the host cell to induce this transformation?
Experiment
You can differentially mark proteins & nucleic acids using radioactive isotopes of
sulfur & phosphorus. These isotopes give off radiation which can be detected.
Proteins were labeled with S35 which made them “glow” – Think Homer Simpson
Nucleic acids were labeled with P32 which made them “glow”
Chapter 12 - DNA Structure & Replication
Chapter 12 - DNA Structure & Replication
Chapter 12 - DNA Structure & Replication
1950 Hershey & Chase
They incubated their host bacteria cells with either S35 labeled viruses or P32 labeled
viruses & here are the results
Both sets of bacteria cells were transformed into cells that produced more of the viruses
In the S35 labeled experiment the radioactivity was localized outside the bacteria cells.
Proteins were not injected into the bacteria cells & the cells were still transformed
In the P32 labeled experiment the radioactivity was localized inside the bacteria cells.
Nucleic acids were injected into the bacteria cells & the cells were transformed
Conclusion: Injection of nucleic acids induces transformation
Chapter 12 - DNA Structure & Replication
Structure of DNA
The basic building block of a DNA molecule is the nucleotide
1.
Deoxyribose – 5 carbon sugar – forms part of the backbone structure
2.
Nitrogenous base – Information is coded in the sequence of these bases
A. Purines – Double ring structure – Adenine (A) & Guanine (G)
B. Pyrimidines – Single ring structure – Thymine (T) & Cytosine (C)
3. Phosphate group – forms part of the backbone structure
Chapter 12 - DNA Structure & Replication
Chapter 12 - DNA Structure & Replication
Chapter 12 - DNA Structure & Replication
Chapter 12 - DNA Structure & Replication
Structure of DNA
Nucleotides are arranged end-to-end in a very specific orientation. The backbone of
the DNA molecule is formed by the phosphate group & the sugar, while the nitrogenous
bases extend perpendicular to the backbone
Each DNA molecule is actually made up of two strands of DNA. The two strands
associate with each other via their nitrogenous bases. Hydrogen bonds form between
them, stabilizing the molecule into its familiar double helix form
Adenine always forms hydrogen bonds with thymine
Guanine always form hydrogen bonds with cytosine
These introduces us to Chargaff’s rule
Regardless of species
1. The number of purines in DNA always equals the number of pyrimidines
2. The amount of adenine always equals the amount of thymine, while the amount of
guanine always equals the amount of cytosine
Chapter 12 - DNA Structure & Replication
Chapter 12 - DNA Structure & Replication
Chapter 12 - DNA Structure & Replication
Structure of DNA
In order for the nitrogenous bases to form these hydrogen bonds, the orientation of the
backbones must be specific.
DNA strands must be anti-parallel to each other, meaning that the sugar-phosphate
backbones must be arranged in opposite directions to each other
The specific directionality is determined by the position of specific carbons & is
referred to as 5’ to 3’
Why is this important?
Important for understanding the intricacies of DNA replication
Why is DNA replication necessary?
When does it occur?
Chapter 12 - DNA Structure & Replication
Chapter 12 - DNA Structure & Replication
Chapter 12 - DNA Structure & Replication
Chapter 12 - DNA Structure & Replication
Overhead diagram of replication – simple version
Chapter 12 - DNA Structure & Replication
DNA Replication
Origin of replication is where it all begins
Helicases bind to the origin & begin unraveling the strands
Single-strand binding proteins keep the strands separated
Topoisomerases relieve tension placed on the DNA molecule caused by this
unraveling, by creating transient cuts which allow the DNA to untwist a little & then
repair the cuts
Once the strands have separated, the strands become templates for replication
Do you remember how the bases are matched up?
New DNA daughter strands cannot be synthesized without some “priming”. As soon
as the parental strands have separated, RNA primase adds a short sequence of RNA
nucleotides to begin the replication process
Once the RNA primer sequence has been laid down, DNA polymerase will begin
adding DNA nucleotides to the new daughter strand of DNA until the strand is
completed
Chapter 12 - DNA Structure & Replication
Chapter 12 - DNA Structure & Replication
Chapter 12 - DNA Structure & Replication
DNA Replication
Since each of the parental strands is used as a template for replication, the process is
referred to as semi-conservative replication
How do we know that this process works in this fashion?
1957 Meselson & Stahl
Chapter 12 - DNA Structure & Replication
Chapter 12 - DNA Structure & Replication
Chapter 12 - DNA Structure & Replication
Chapter 12 - DNA Structure & Replication
Chapter 12 - DNA Structure & Replication
DNA Replication – DETAILS!
DNA replication must occur in a specific direction: 5’ to 3’
Since DNA replication must occur in this direction, a problem arises due to the
antiparallelism of the parental strands
DNA replication is continuous on one parental strand & discontinuous on the other
DNA polymerase reads & lays down the new DNA on the daughter strand
DNA ligase is important for joining the discontinuous DNA fragments on the “lagging”
daughter strand & well as the entire DNA molecule as you will see
Eukaryotic DNAs have multiple origins of replication which will reduce the time it
takes for replication to occur
Chapter 12 - DNA Structure & Replication
Overhead diagram of replication – leading & lagging strand version
Chapter 12 - DNA Structure & Replication
Chapter 12 - DNA Structure & Replication
Chapter 12 - DNA Structure & Replication
PRACTICE QUESTIONS
1.
How did the following individuals contribute to our knowledge of the importance
of DNA as the hereditary material: Miescher, Griffith, Avery et al, Hershey &
Chase?
2.
What is Chargaff’s rule?
3.
What 3 components make up a DNA nucleotide? RNA nucleotide?
4.
What is the difference between DNA & RNA?
5.
What enzymes are used during the steps of replication?