Download DNA structure and replication notes

Honors Biology
 1. Discovery
of Genetic Material
 2. DNA Structure
 3. The Race to Solve the Puzzle of DNA
Structure
 4.DNA Replication
•
Biologists knew that genes are located on
chromosomes (made of DNA and protein)
– DNA and protein were the candidates for the
genetic material
– Until the 1940s, the case for proteins seemed
stronger because proteins appeared to be more
structurally complex and functionally specific.
– Biologists finally established the role of DNA in
heredity through studies involving bacteria and
the viruses that infect them.
•
•
1928; British medical officer
Griffith was studying two strains of a
bacterium:
– a pathogenic (disease-causing) strain that cause
pneumonia
– a harmless strain.
•
Found that when he mixed a dead version
of the pathogenic bacteria and harmless
bacteria, some living bacterial cells were
converted to the disease-causing form.
– Furthermore, all of the descendants of the
transformed bacteria inherited the newly
acquired ability to cause disease.
 Clearly, some
chemical component of the
dead bacteria could act as a
“transforming factor” that brought about
a heritable change.



1952; American biologists
Experiments showed that DNA is the genetic material
of a virus (bacteriophage or phage, for short) called T2,
which infects E.coli
T2 consists solely of DNA and protein; DNA-containing
head and a hollow tail with six jointed fibers extending
from it.

T2 infects bacteria by attaching to the surface with its
fibers and injecting its hereditary material.
• Raised the question: Is it DNA being passed on
or protein?
 To
answer protein or DNA question, they
devised an experiment to determine
what kinds of molecules the phage
transferred to E.coli during infection

Used a few relatively simple tools:
• Chemicals containing radioactive isotopes
 To label the DNA and protein in T2
 Used radioactive sulfur and phosphorous
 Sulfur is in proteins. Phosphorous is in DNA.
• A radioactivity detector
• A kitchen blender
• And a centrifuge (device that spins test tubes to
separate particles of different weights.
 The
1.
2.
3.
4.
5.
6.
Experiment
First they grew T2 with E.Coli with radioactive sulfur.
Then, they grew a separate batch of phages in a solution
containing radioactive phosphorous.
They allowed the two batches of T2 to infect separate samples of
nonradioactive bacteria
Shortly after the onset of infection, they agitated the cultures in
a blended to shake loose any parts of the phages that remained
outside the bacterial cells.
They then spun the mixtures in a centrifuge. The cells were
deposited as a pellet at the bottom of the centrifuge tubes, but
phages and parts of phages being lighter, remained suspended
in the liquid.
The researchers then measured the radioactivity in the pellet
and the liquid.

The Results
• Found that when the bacteria has been infected with
T2 phages containing labeled protein, the
radioactivity ended up in the liquid but not bacteria.
 Result suggested that the phage protein did not enter the
cells.
• But when the bacteria had been infected with
phages whose DNA was tagged, then most of the
radioactivity was in the pellet, made up of bacteria.
 When these bacteria were returned to liquid growth
medium, they soon died and lysed and released new
phages that contained radioactive phosphorous in their
DNA but no radioactive sulfur.
 The
Conclusions
• They concluded that T2 injects its DNA into the host
cell, leaving virtually all its protein outside.
• They demonstrated that it is the injected DNA
molecules that cause cells to produce additional
phage DNA and proteins, making new complete
phages.
• This indicated that DNA contained the instructions
for making proteins
• ***THESE RESULTS CONVINCED MOST SCIENTISTS
THAT DNA IS THE HEREDITARY MATERIAL!!!***
 Deoxyribonucleic
Acid (DNA)
• Monomers made up of nucleotides:
 Nucleotides consist of:
 A five carbon sugar, deoxyribose
 Phosphate group
 Nitrogenous base (Adenine, Guanine, Cytosine, Thymine)
• Double helix consists of:
 Sugar-phosphate backbone held by covalent bonds
 Nitrogen bases are hydrogen bonded together; A pairs with T and C
pairs with G
 Nucleotides consist of:
 A five carbon sugar, deoxyribose
 Four in it’s ring, one extending above the ring
 Missing one oxygen when compared to ribose
 Phosphate group
 Is the source of the “acid” in nucleic acid
 Nitrogenous base (Adenine, Guanine, Cytosine, Thymine)
 A ring consisting of nitrogen and carbon atoms with various
functional groups attached
 Double ring= purines (A and G)
 Single ring= pyrimidines (T and C)
 DNA’s
sugar-phosphate backbones run in
opposite directions.
 Each strands has a 3’ end and a 5’ end.
 The primed number is referring to the
carbon atoms of the nucleotide sugars.
 At one end of each DNA strand, the
sugar’s 3’ carbon atom is attached to an –
OH group, at the other end, the sugar’s 5’
carbon has a phosphate group.
A
few scientists working on the puzzle
trying to determine the 3-D structure:
Franklin, Watson and Crick
 Rosalind Franklin observed an X-ray
crystallography image of the basic shape
of DNA
 Watson
saw this image, and with just a
glance deduced the basic shape of DNA
to be a helix with a uniform diameter of 2
nm, with its nitrogenous bases stacked
about one-third of a nanometer apart.
• The diameter of the helix suggested it was made
up of two polynucleotide strands=> DOUBLE
HELIX!
 Watson
and Crick began trying to
construct a double helix that would
conform both Franklin’s data and what
was currently known about the chemistry
of DNA.
 Franklin had concluded that the sugarphosphate backbones must be on the
outside of the double-helix, forcing the
nitrogenous bases to swivel to the
interior of the molecule.
 Watson
and Crick found that Adenine
always paired with Thymine, and
Guanine and Cytosine, to ensure a
uniform diameter.
 Complementary base pairing was
explained both by the physical attributes
and chemical bonding of DNA, along with
data obtained by Chargaff
 Chargaff’s
rules: A always pairs with T
and G always pairs with C.
 Only apply to base pairing, not the
sequence of nucleotides
• The sequence of bases can vary in countless
ways, and each gene has a unique order of
nucleotides, or base sequence
 1962, Watson
and Crick received the
Nobel Prize for their work (Franklin
would have received it as well, but she
died from cancer in 1958; Nobel Prizes
are never awarded to the deceased)
“It has not escaped our notice that the specific pairing we have
postulated immediately suggests a possible copying mechanism
for the genetic material”
~Watson and Crick
 Logic
behinds Watson-Crick’s proposal
for how DNA is copied
• Can be seen by covering one of the strands in
the parental DNA molecule with a piece of
paper: you can determine the bases of the
covered strand by applying the base-pairing
rules: A pair with T, and G pairs with C.
• Watson and Crick predicted that a cell applies
the same rules when copying its genes.

Figure 10.4A Template model for DNA
replication
1. First, the two strands of parental DNA separate,
and each becomes a template for the assembly
of a complementary strand from a supply of free
nucleotides.
2. The nucleotides line up one at a time along the
template strand in accordance with the basepairing rules
3. Enzymes link the nucleotides to form the new
DNA strands.
4. Completed new molecules, identical to the
parental molecule, are known as daughter DNA.
 Semi-conservative
model
• Watson and Crick’s model predicts that when a
double helix replicates, each of the two daughter
molecules will have one old strand, which was
part of the parental molecule, and one new made
strand.
• Known as semi-conservative model because half
of the parental molecule is maintained
(conserved) in each daughter molecule.
• Confirmed by experiments performed in the
1950s.
 The
opposite orientation of the strands is
important in DNA replication.
 DNA polymerases link DNA nucleotides
to a growing daughter strand, only to the
3’ end of the strand, never to the 5’ end.
 Thus, a daughter DNA strand can only
grow in the 5’3’ direction.
 We
will be looking at three steps in DNA
Repication:
• 1. Initiation
• 2. Elongation
• 3. Termination
Initiation:
• DNA replication begins at specific sites on
the double helix referred to as origins of
replication.
• An enzyme called helicase binds to DNA
and separates the strands.
 Uses energy from ATP
 Single-strand binding protein (SSB) binds to
each strand to prevent reannealing
• Initiation (continued)
 Replication proceeds in both directions , creating
replication “bubbles”
 DNA has many origins of replication that can start
simultaneously, for time efficiency.
 Thousands of bubbles can be present, and eventually
all the bubbles merge, yielding two completed
daughter DNA molecules.
 Elongation:

Replication fork forms:
• Partial opening of a DNA helix to form two single
strands that has a fork appearance.
• Primers , made of short stetches of RNA, are required
by DNA polymerases during replication
 synthesized by an enzyme called RNA primase.
• Elongation (continued):
• DNA polymerase removes primers and adds DNA
nucleotides.
 We will be looking at two types of DNA Polymerase:
DNA polymerase III and DNA polymerase I
• Elongation (continued):
• one of the daughter strands can be synthesized
in one continuous fashion from an initial primer,
working toward the forking point of the parental
DNA. leading strand
 Only a single priming event is required, and then the
strand can be extended indefinitely by DNA
polymerase III.
Elongation (continued):
• The other daughter strand polymerase molecules
must work outward from the forking point, is
synthesized in short pieces as the fork opens up in a
discontinous fashion involving multiple priming
events. lagging strand
 Elongation
(continued):
 Lagging strand (continued)
 DNA Polymerase III adds nucleotides in 5’3’
direction. Primers get removed via and replaced
by DNA via DNA polymerase I
 Fragments formed are called Okazaki fragments
 DNA ligase links (ligates) the pieces together into a
single DNA strand.
 Termination:
• At the completion DNA replication, you end up
with two identical strands of DNA.
• Each DNA molecule contains one original parent
strand, and one new daughter strand. Semi
conservative replication
 http://www.wiley.com/college/pratt/047
1393878/student/animations/dna_replica
tion/index.html
 Key
enzymes:
• Helicase: unwinds the double helix
• Primase: synthesizes RNA primers
• SSB: stabilizes single-stranded regions; prevents
reannealing
• DNA polymerase III- synthesized DNA
• DNA polymerase I- erases primer and fills gaps
• DNA ligase- joins the ends of DNA segments;
DNA repair


DNA polymerase carry out a proofreading step that
quickly removes nucleotides that have base-paired
incorrectly
DNA polymerases and DNA ligase are also involved in
repairing damaged DNA by harmful radiation or toxic
chemicals
• Process is not only fast but also amazingly accurate
• Typically, only about one DNA nucleotide per billion is
incorrectly paired
• Ensure that all somatic cells in a multicellular organism carry
the same genetic information.