We have briefly discussed that DNA is the genetic molecule of life. In eukaryotic organisms DNA (along with special proteins) is found in chromosomes. All cell activities are controlled by DNA.

We also know that the metabolic activities of a cell are all catalyzed by enzymes, specific proteins, and that the instructions for the synthesis of proteins are found in the structure of DNA.

To continue with our knowledge, a gene is a functional region of DNA that specifies a certain inheritable characteristic or trait. This region of DNA stores the information that specifies the sequence of amino acids that form a specific polypeptide. The genes we inherit from our parents determine the polypeptides we synthesize in our cells, which determine the structure and functioning of our cells and tissues.

What DNA is and how DNA works is the subject of this unit of AP Biology . We will look at the structure and functions of DNA, how the information stored in DNA is used to direct cell activities and how cells regulate the activity of their genetic molecules.

When we study the process of cell reproduction and mitosis, we will learn that DNA molecules (chromosomes) are precisely duplicated prior to cell division to ensure that all cells of an individual have exactly the same DNA. It is the process of mitosis that distributes the duplicated chromosomes of the original cell equally into the new cells.

The search for the molecule of inheritance spanned a century from the mid-1850's to 1953, when Francis Crick and James Watson announced they had determined the three dimensional structure of DNA. The steps to this discovery are a good example of the process of science.

DNA was first isolated by Meischer in the mid-1800's. He identified a phosphorus containing acid found in the nuclei of cells. About the same time Feulgen developed a stain that was selective for this material of the nucleus. Fuelgen noted that the volume of the nuclear material was the same for all body (somatic) cells, but gametes had half as much of this material. He also noted that cells that were about to divide had twice as much nuclear material. No one knew how to interpret this information and nothing much happened in molecular genetics for the next fifty years.

Although genes, chromosomes and the transmission of genetic information were studied extensively in the first half of the 20^th century, the molecular structure of a gene was not known. For most of this period of time, scientists believed that the genetic molecule had to be protein ذbecause of protein's diversity of structures and specificity of functions. In contrast, DNA is composed of some fairly simple molecules: phosphates, a five-carbon sugar, and four different nucleotides, so the means by which it could serve as the genetic molecule was perplexing.

Discovering the Genetic Molecule
Evidence #1
In 1928, Fred Griffith was trying to find a vaccine to protect against a pneumonia-causing bacterium, Streptococcus pneumoniae. He isolated two strains of the bacterium. One had a polysaccharide capsule which gave it a smooth (S) appearance in culture. The other form appeared rough (R) in culture. The S form is a virulent form of the bacterium, since the capsule protects it from harmful things in its environment, which in this case is the immune system of the host.

Griffith injected bacteria into mice, and observed what happened. Mice injected with S forms died. Mice injected wit R forms lived. Mice injected with heat-killed S forms lived. But: Mice injected with a mixture of heat-killed S forms and live R forms died, and when necropsied, contained live S form bacteria.

What did this mean?

1. he production of a capsule is an inheritable trait that distinguishes the R form from the S form of the bacterium.
   
2. Somehow, the heat which killed the S cells did not damage the material that had genetic instructions so that this material (instructions on how to make a capsule) could be incorporated into the living R cells (The R cells could pick up this material from the environment) transforming these R cells into virulent S forms.
   
3. Griffith called this the Transformation Principle.

   Today, transformation is defined as the process by which external DNA is assimilated into a cell changing its genotype and phenotype. Transformation is one of the processes used in DNA technologies.
   

   Evidence #2
   Starting in the 1930's, a group of microbiologists, headed by Oswald Avery, repeated Griffith's experiments adding a series of enzymes (from the pancreas) that selectively destroyed DNA, RNA or protein.

   They performed the following experiments:

   1. Mice + Protein-digesting enzyme + heat-killed S + R --> Dead Mice
   2. Mice + RNA-digesting enzyme + heat-killed S + R ----> Dead Mice
   3. Mice + DNA-digesting enzyme + heat-killed S + R ----> Live Mice
   
   In 1944, Avery concluded that DNA was the genetic molecule. Transformation was prevented only when DNA was destroyed. Many scientists still disputed this conclusion, since the structure of DNA was not known, and Avery could not say how DNA might work.

   Evidence # 3
   Bacteriophages (viruses that invade bacteria and convert the bacteria into virus making machines) proved to be the means by which the question was finally answered. In 1952, Hershey and Chase (and others) confirmed that DNA was the genetic molecule. Viruses have just DNA (or sometimes just RNA) and a protein coat. Proteins contain sulfur, but not phosphorus and DNA contains phosphorus, but not sulfur.

   Hershey and Chase used radioactive Sulfur and Phosphorus to "label" T2 phages. They then tracked the invasion of phages into host bacteria (a strain of E coli) and what part of the new generation phages became radioactive. Since only the DNA of the new generation of phages was radioactive, Hershey and Chase were able to confirm that DNA was the genetic molecule.

   Still, the structure of DNA was unknown, so no one had an explanation for how DNA could do its job. The search continued.
   
   Structural Evidences supporting DNA as the Genetic Molecule
   Demonstrating that DNA was the genetic molecule was one significant part of the solution. To know how DNA works also required knowledge of the three dimensional structure of the molecule.

   By the early 1950's the following was known.
   

   1. DNA was composed of nucleotides. Each nucleotide contained:
      
      • Phosphate (P)
        
      • The 5-carbon sugar, deoxyribose
        
      • One of four different nitrogen-containing bases
        Two were double ring purines
        
        Adenine
        Guanine
        
        Two were single ring pyrimidines
        
        Thymine
        Cytosine
        

        
        

      • The sugar phosphate formed a backbone with one of the four bases attached to the side of the sugar.

      • Long chains of nucleotides could be formed linking sugar-phosphate backbones with the Nitrogen bases attached to the side of the sugar molecules (S-P-S-P-S-P-S-P, etc.).

        Specifically, the phosphate bonded to the 5' carbon of the sugar molecule, leaving the 3' carbon of the sugar to attach to the next phosphate. The nitrogen base attached to the 1' carbon of the sugar molecule. This little detail is important to the structure of DNA. Deoxyribose is a 5-carbon sugar. In a carbon compound, each of the carbons is given a number. Deoxyribose is a 5-carbon sugar. Who bonds to what carbon is critical to DNA's structure.

   2. Mirsky restated work from the 1850's that determined the relationship of the volume of DNA in the nucleus for normal body cells, cells just prior to division and in gametes. This provided evidence that DNA was the genetic molecule because it corresponded with the behavior of chromosomes in mitosis and meiosis.

      
      

   3. Erwin Chargaff's work in 1947:
      • The four nitrogen bases were not present in equal amounts
        
      • The amounts differed in different species
        
      • But
        
        
        • The amount of Adenine was always the same as Thymine
          
        • The amount of guanine was always the same as Cytosine

          This information is known as Chargaff's rules

   4. X-ray diffraction (best done by Rosalind Franklin at King's College in London) showed that DNA:
      • was long and thin
        
      • had a uniform diameter of 2 nanometers
        
      • had a highly repetitive structure
        
      • was probably helical in shape, like a circular stairway

        
        

   Watson and Crick
   From this information, Watson and Crick determined the structure of DNA in 1953 and published their work in Nature. They surmised (and confirmed):
   
   • DNA was probably a double strand (because of the 2 nm diameter)*

     This was interesting because Linus Pauling, who was also working on the structure of DNA, had just written a proposal for DNA having a triple stranded structure.

   
   
   • To maintain the uniform diameter, a double-ring base probably would pair with a single-ring base along the length of the molecule

     Adenine can hydrogen bond to thymine at 2 places.
     Guanine can hydrogen bond to cytosine at 3 places

   
   
   • This explained Chargaff's findings that the amounts of adenine and thymine were the same, and the amounts of guanine and cytosine were the same for a species.

   • Two strands of nucleotides, with their bases hydrogen-bonded to each other would form a ladder, if and when the sugar phosphate backbones ran in opposite directions to each other, or anti-parallel to each other, and twisted to form a double helix. This is where that 5' and 3' bonding just mentioned gets important. The end of a DNA molecule will have a free sugar (3'end) on one side and a free phosphate (5' end) on the other side.
     

   The constancy of the complementary base-pairing is critical to the structure of DNA. DNA of different species and of different genes shows variation in the sequence of base pairs in the DNA chain (which base pair follows the next).

   Once the structure of DNA was determined, active research could take place in how DNA can duplicate (or replicate) prior to cell division and in how DNA stores genetic information. We shall look at both in this unit, but just the mechanism of DNA replication in this chapter.

   DNA Replication
   

   • DNA is a double stranded molecule. The two chains (or strands) are attached by hydrogen bonds between the nitrogen bases.
     
   • The two strands are anti-parallel to each other (run in opposite directions). That is, the 3' carbon of the sugar (the free sugar end) starts one strand and the 5' carbon sugar end (the free phosphate end) starts the other. This is necessary for the base-pairs to hydrogen bond correctly.
     
   • Adenine must bond to thymine, and guanine must bond to cytosine.
   Because of the complementary base pairing, if one side of the double stranded molecule is known, we automatically know what the other half is. This model for DNA replication is known as the semi-conservative model for DNA replication.
   
   The process of DNA Replication (or Duplication)
   There are three basic steps to DNA replication:
   
   • The two DNA strands of the parental chromosome must unwind and separate.
     
   • Each strand of the parent chromosome serves as a template for the synthesis of a daughter strand. DNA is always synthesized in the 5' --> 3' direction from the 3' --> 5' template.
     
   • The newly synthesized double helix of each parent-daughter combination rewinds to form the DNA chains of a replicated chromosome. Each new DNA molecule is composed of one-half of the parent chromosome and one-half newly synthesized DNA.

   A few details about the process:
   

   • Prior to cell division, the enzyme, DNA helicase, facilitates the unwinding of the double-stranded DNA molecule forming replication bubbles in the DNA molecule.

   • Replication forks are formed at the origin of each bubble. New DNA is replicated behind each fork as it progresses along the DNA molecule in both directions from the origin.

   • In eukaryotic organisms, there are many, many replication units involved in the replication of DNA on each chromosome. Each replication unit forms a replication fork at its "origin". As DNA replication progresses, replication units join other units when they meet an adjacent replication forks.

   • Each of the two strands of the DNA molecule in the fork serves as a template for the attachment of its complement nucleotides (A-T, C-G, G-C and T-A). This takes place on both sides of the replication forks simultaneously, but in opposite directions.

   • The enzyme, DNA polymerase, promotes the synthesis of the new DNA strands, by recognizing the appropriate complementary base needed and by bonding appropriate daughter nucleotides to the growing DNA molecule.

   • Unfortunately, DNA polymerase has two problems: it can only work in one direction, and it cannot get started on a single chain DNA strand.

     DNA polymerase can only attach a nucleotide to the exposed -OH group on the 3' end of the leading strand template. DNA is always synthesized in the 5' --> 3' direction from the 3' --> 5' template. This sounds stranger than it really is. Since the chains of DNA are antiparallel, or opposite, the new nucleotides attached will be in a 5' to 3' direction, while it is adding new nucleotides at the 3' template end.

     This is fine for one of the two DNA strands of the original molecule, but not for the second strand, which is running in the opposite direction.

     The upper 3' end of the original DNA molecule is called the leading strand of the template because replication starts at that point.

   • DNA polymerase's second problem is that it cannot get started by itself; it needs to add to a pre-existing chain. This is solved by starting replication with a RNA primer activated by an enzyme called primase. Primase catalyzes a short RNA molecule or primer that is used to start the synthesis of DNA.

   • Once the DNA strand has been "primed" by primase adding the RNA primer, DNA polymerase can go to work adding nucleotides to the 3' open end of the newly forming molecule. DNA polymerase will also eventually replace the RNA nucleotides of the primer with DNA nucleotides.

   • DNA polymerase works by positioning the parent strand of the DNA molecule into a groove and pulls the DNA through the groove as it directs the synthesis of new DNA.
     
     Attaching Nucleotides in DNA Replication

   • DNA replication is continuous along one side (the leading strand) of the original template DNA molecule, because the newly synthesized daughter nucleotides can follow the path of DNA helicase.

   • However, the other template strand of DNA is unzipped in the 5' to 3" direction so new DNA synthesis must be discontinuous. This DNA strand is called the lagging strand, because its rate of synthesis lags behind that of the leading strand. Additional DNA polymerase enzymes must be attached at unzipped regions, but the synthesis direction of the lagging strand is opposite the direction of the unzipping DNA helicase enzyme.

     New DNA polymerase enzyme molecules have to attach and work on small portions of the lagging strand of the unzipped DNA molecule. Moreover, the lagging strand must be looped to provide the needed orientation for DNA polymerase.

     The enzyme, DNA ligase, links the short pieces, called Okazaki fragments, of the lagging side together. Note: Each Okazaki fragment must be initiated by a RNA primer.

   • After the DNA is replicated along its entire length, two DNA molecules have been formed, each half the original and half new nucleotides. Both DNA molecules are identical to each other and to the original.

     Proofreading the DNA
     DNA errors occur about 1 in 1 billion nucleotides in the final DNA, but pairing errors do occur during the process as often as 1 per 10,000 pairs. As you might expect, DNA is proofread by DNA polymerase as it is being replicated. If there is an error, it deletes the mistake, and replaces it with the correct nucleotide.

     Damage to DNA molecules occurs daily by exposure to routine molecules in the environment, and even to normal body temperatures. DNA repairing enzymes are always at work. Even so, some mistakes do not get repaired. Deterioration of DNA replication accuracy is likely a contributing factor in aging.