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 Experiment1. First they grew T2 with E.Coli with radioactive sulfur.2. Then, they grew a separate batch of phages in a solution

containing radioactive phosphorous. 3. They allowed the two batches of T2 to infect separate

samples of nonradioactive bacteria4. 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.

5. 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.

6. 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 sugar-phosphate 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 base-pairing 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/0471393878/student/animations/dna_replication/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.