A. DNA as the Genetic Material
1. The search for genetic material led to DNA.
a. In the 1940s it was already known that genes were on chromosomes and that they were made of DNA but it was not known how the DNA molecule was constructed.
b. By 1947, Erwin Chargaff had developed a series of rules based on a survey of DNA composition in different organisms.
1. He already knew that DNA was a polymer of nucleotides consisting of a nitrogenous base, deoxyribose, and a phosphate group.
2. The bases could be adenine (A), thymine (T), guanine (G), or cytosine (C).
3. Chargaff noted that the DNA composition varies from species to species. In any one species, the four bases are found in characteristic, but not necessarily equal, ratios.
a. Human DNA is 30.9% adenine, 29.4% thymine, 19.9% guanine and 19.8% cytosine.
4. He also found a regularity in the ratios of nucleotide bases which are known as Chargaff�s rules.
a. The number of adenines was approximately equal to the number of thymines (%T = %A).
b. The number of guanines was approximately equal to the number of cytosines (%G = %C).
5. Chargaff did not know that the reason the amounts of A and T and G and C were equal was because they were paired in the DNA molecule. This was discovered by James Watson and Francis Crick.
2. Watson and Crick discovered the double helix by building models to conform to X-ray data.
a. By the beginning of the 1950s, researchers were focusing on understanding the three-dimensional structure of DNA.
b. Maurice Wilkins and Rosalind Franklin used X-ray crystallography to study the structure of DNA.
1. In this technique, X-rays are diffracted as they pass through aligned fibers of purified DNA.
2. The diffraction pattern can be used to deduce the three-dimensional shape of molecules. (Fig. 16.4)
c. James Watson concluded from their research that DNA was helical in shape and he deduced the width of the helix and the spacing of bases.
1. The width of the helix indicated it was made up of two strands and Watson and his colleague Francis Crick began to work on a model of DNA with two strands, the double helix.
2. Using molecular models made of wire, they first tried to place the sugar-phosphate chains on the inside. (Fig. 16.3)
3. However, this did not fit the X-ray measurements and other information on the chemistry of DNA.
4. The key breakthrough came when Watson put the sugar-phosphate chains on the outside and the nitrogen bases on the inside of the double helix.
5. The sugar-phosphate chains of each strand are like the side ropes of a rope ladder.
6. Pairs of nitrogen bases, one from each strand, form rungs.
The ladder forms a twist every ten bases. (Fig. 16.5)
7. The nitrogenous bases are paired in specific combinations: adenine with thymine and guanine with cytosine.
8. Pairing like nucleotides did not fit the uniform diameter indicated by the X-ray data.
a. A purine-purine pair would be too wide and a pyrimidine-pyrimidine pairing would be too short.
b. Only a pyrimidine-purine pairing would produce the 2nm diameter indicated by the X-ray data.
9. In addition, Watson and Crick determined that chemical side groups of the nitrogen bases would form hydrogen bonds, connecting the two strands. (Fig. 16.6)
a. Based on details of their structure, adenine would form two hydrogen bonds only with thymine and guanine would form three hydrogen bonds only with cytosine.
b. This finding explained Chargaff�s rules.
d. The base-pairing rules dictate the combinations of nitrogenous bases that form the "rungs" of DNA.
1. However, this does not restrict the sequence of nucleotides along the DNA strand.
2. The linear sequence of the four bases can be varied.
3. Each gene has a unique order of nucleotides.
e. In April 1953, Watson and Crick published a one-page paper in Nature reporting their double helix model of DNA.
f. In 1962 Watson and Crick received the Nobel Prize for the discovery of the double helical structure of DNA. Franklin could not share in prize because she died in the late 1950s from cancer due to X-ray exposure. The Nobel prize is not awarded posthumously.
B. DNA Replication and Repair
1. During DNA replication, base pairing enables existing DNA strands to serve as templates for new complementary strands.
a. In a second paper Watson and Crick published their hypothesis for how DNA replicates.
1. Essentially, because each strand is complementary to the other, each can form a template when separated.
2. The order of bases on one strand can be used to add in complementary bases and therefore duplicate the pairs of bases exactly.
b. Experiments in the late 1950s by Matthew Meselson and Franklin Stahl supported the semiconservative replication model, proposed by Watson and Crick.
1. When a cell copies a DNA molecule, each strand serves as a template for ordering nucleotides into a new complementary strand. (Fig. 16.7)
2. A large team of enzymes and other proteins carries out DNA replication
a. Overview (Textbook Activity 16D)
1. It takes E. coli less than an hour to copy each of the 5 million base pairs in its single chromosome and divide to form two identical daughter cells.
2. A human cell can copy its 6 billion base pairs and divide into daughter cells in only a few hours.
3. This process is remarkably accurate, with only one error per billion nucleotides.
4. More than a dozen enzymes and other proteins participate in DNA replication.
b. Origins of replication (Fig. 16.10)
1. In eukaryotes, there may be hundreds or thousands of origins of replication per chromosome.
2. At the origin sites, enzymes separate the DNA strands, forming a replication "bubble" with replication forks at each end.
3. The replication bubbles elongate as the DNA is replicated and eventually fuse.
c. Elongating a new strand
1. DNA polymerase III catalyzes the elongation of new DNA at a replication fork.
2. As nucleotides align with complementary bases along the template strand, they are added to the growing end of the new strand by the polymerase.
3. The rate of elongation is about 500 nucleotides per second in bacteria and 50 per second in human cells.
d. How does DNA polymerase III work?
1. The strands in the double helix are antiparallel (the sugar-phosphate backbones run in opposite directions). (Fig. 16.12)
2. Each DNA strand has a 3� end with a free hydroxyl group attached to deoxyribose and a 5� end with a free phosphate group attached to deoxyribose.
3. The 5� -> 3� direction of one strand runs counter to the 3� -> 5� direction of the other strand.
4. DNA polymerase can only add nucleotides to the free 3� end of a growing DNA strand. (Fig. 16.13)
5. A new DNA strand can only elongate in the 5�->3� direction.
6. In the replication bubble, one parental strand is oriented 3�->5�, while the other antiparallel parental strand is oriented 5�->3�.
7. At the replication fork, one parental strand (3�-> 5� into the fork), can be used by DNA polymerase as a template for a continuous complementary strand, the leading strand.
8. The other parental strand (5�->3� into the fork), is copied away from the fork in short segments (Okazaki fragments) that make up the lagging strand.
9. Okazaki fragments, each about 100-200 nucleotides, are joined by DNA ligase to form the sugar-phosphate backbone of a single DNA strand.
e. Priming DNA synthesis (Fig. 16.14)
1. DNA polymerase cannot initiate synthesis of a polynucleotide because it can only add nucleotides to the end of an existing chain.
2. To start a new chain requires a primer, a short segment of RNA.
3. The primer is about 10 nucleotides long in eukaryotes.
4. Primase, an RNA polymerase, links ribonucleotides that are complementary to the DNA template into the primer.
5. After formation of the primer, DNA polymerase can add deoxyribonucleotides to the 3� end of the ribonucleotide chain.
6. Another DNA polymerase, DNA polymerase I, later replaces the primer ribonucleotides with deoxyribonucleotides complementary to the template.
7. The leading strand requires the formation of only a single primer as the replication fork continues to separate.
8. For the lagging strand, each Okazaki fragment requires a primer. (Fig. 16.16)
9. After the primer is formed, DNA polymerase can add new nucleotides away from the fork until it runs into the previous Okazaki fragment.
10. The primers are converted to DNA before DNA ligase joins the fragments together.
f. Other proteins in DNA replication (Fig. 16.15)
1. Helicase untwists and separates the template DNA strands at the replication fork.
2. Single-strand binding proteins keep the unpaired template strands apart during replication. (Textbook Activity 16 E)
3. Enzymes proofread DNA during its replication and repair damage in existing DNA.
a. Mistakes during the initial pairing of template nucleotides and complementary nucleotides occur at a rate of one error per 10,000 base pairs.
1. DNA polymerase III proofreads each new nucleotide against the template nucleotide as soon as it is added.
2. If there is an incorrect pairing, the enzyme removes the wrong nucleotide and then resumes synthesis.
3. The final error rate is only one per billion nucleotides.
b. Mismatched nucleotides that are missed by DNA polymerase or mutations that occur after DNA synthesis is completed can often be repaired.
c. DNA molecules are constantly subject to potentially harmful chemical and physical agents.
1. Reactive chemicals, radioactive emissions, X-rays, and ultraviolet light can change nucleotides in ways that can affect encoded genetic information.
2. DNA bases often undergo spontaneous chemical changes under normal cellular conditions.
d. Each cell continually monitors and repairs its genetic material, with over 130 repair enzymes identified in humans.
1. In mismatch repair, enzymes fix incorrectly paired nucleotides.
2. In nucleotide excision repair, a nuclease cuts out a segment of a damaged strand. (Fig. 16.17)
a. The gap is filled in by DNA polymerase and ligase.
b. Thymine dimers are created by exposure to U.V. light.