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Lecture Chapter Outline
The Molecular Basis of Inheritance
Overview: Life's Operating Instructions In April 1953, James Watson and Francis Crick shook the scientific world with an elegant double-helical model for the structure of deoxyribonucleic acid, or DNA. Your genetic endowment is the DNA you inherited from your parents. Nucleic acids are unique in their ability to direct their own replication. The resemblance of offspring to their parents depends on the precise replication of DNA and its transmission from one generation to the next. It is this DNA program that directs the development of your biochemical, anatomical, physiological, and (to some extent) behavioral traits. Concept 16.1 DNA is the genetic material
The search for genetic material led to DNA.
Once T. H. Morgan's group showed that genes are located on chromosomes, the two constituents of chromosomes--proteins and DNA--were the candidates for the genetic material. Until the 1940s, the great heterogeneity and specificity of function of proteins seemed to indicate that proteins were the genetic material. However, this was not consistent with experiments with microorganisms, such as bacteria and viruses. The discovery of the genetic role of DNA began with research by Frederick Griffith in 1928. He studied Streptococcus pneumoniae, a bacterium that causes pneumonia in mammals. One strain, the R strain, was harmless. The other strain, the S strain, was pathogenic. Griffith mixed heat-killed S strain with live R strain bacteria and injected this into a mouse. The mouse died, and he recovered the pathogenic strain from the mouse's blood. Griffith called this phenomenon transformation, a phenomenon now defined as a change in genotype and phenotype due to the assimilation of foreign DNA by a cell.
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For the next 14 years, scientists tried to identify the transforming substance. Finally in 1944, Oswald Avery, Maclyn McCarty, and Colin MacLeod announced that the transforming substance was DNA. Still, many biologists were skeptical. Proteins were considered better candidates for the genetic material. There was also a belief that the genes of bacteria could not be similar in composition and function to those of more complex organisms. Further evidence that DNA was the genetic material was derived from studies that tracked the infection of bacteria by viruses. Viruses consist of DNA (or sometimes RNA) enclosed by a protective coat of protein. To replicate, a virus infects a host cell and takes over the cell's metabolic machinery. Viruses that specifically attack bacteria are called bacteriophages or just phages. In 1952, Alfred Hershey and Martha Chase showed that DNA was the genetic material of the phage T2. The T2 phage, consisting almost entirely of DNA and protein, attacks Escherichia coli (E. coli), a common intestinal bacteria of mammals. This phage can quickly turn an E. coli cell into a T2-producing factory that releases phages when the cell ruptures. To determine the source of genetic material in the phage, Hershey and Chase designed an experiment in which they could label protein or DNA and then track which entered the E. coli cell during infection. They grew one batch of T2 phage in the presence of radioactive sulfur, marking the proteins but not DNA. They grew another batch in the presence of radioactive phosphorus, marking the DNA but not proteins. They allowed each batch to infect separate E. coli cultures. Shortly after the onset of infection, they spun the cultured infected cells in a blender, shaking loose any parts of the phage that remained outside the bacteria. The mixtures were spun in a centrifuge, which separated the heavier bacterial cells in the pellet from lighter free phages and parts of phage in the liquid supernatant. They then tested the pellet and supernatant of the separate treatments for the presence of radioactivity. Hershey and Chase found that when the bacteria had been infected with T2 phages that contained radiolabeled proteins,
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most of the radioactivity was in the supernatant that contained phage particles, not in the pellet with the bacteria. When they examined the bacterial cultures with T2 phage that had radiolabeled DNA, most of the radioactivity was in the pellet with the bacteria. Hershey and Chase concluded that the injected DNA of the phage provides the genetic information that makes the infected cells produce new viral DNA and proteins to assemble into new viruses. The fact that cells double the amount of DNA in a cell prior to mitosis and then distribute the DNA equally to each daughter cell provided some circumstantial evidence that DNA was the genetic material in eukaryotes. Similar circumstantial evidence came from the observation that diploid sets of chromosomes have twice as much DNA as the haploid sets in gametes of the same organism. By 1947, Erwin Chargaff had developed a series of rules based on a survey of DNA composition in organisms. He already knew that DNA was a polymer of nucleotides consisting of a nitrogenous base, deoxyribose, and a phosphate group. The bases could be adenine (A), thymine (T), guanine (G), or cytosine (C). 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. He also found a peculiar regularity in the ratios of nucleotide bases that are known as Chargaff's rules. In all organisms, the number of adenines was approximately equal to the number of thymines (%T = %A). The number of guanines was approximately equal to the number of cytosines (%G = %C). Human DNA is 30.9% adenine, 29.4% thymine, 19.9% guanine, and 19.8% cytosine. The basis for these rules remained unexplained until the discovery of the double helix.
Watson and Crick discovered the double helix by building models to conform to X-ray data.
By the beginnings of the 1950s, the race was on to move from the structure of a single DNA strand to the three-dimensional structure of DNA. Among the scientists working on the problem were Linus Pauling in California and Maurice Wilkins and Rosalind Franklin in London.
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Maurice Wilkins and Rosalind Franklin used X-ray crystallography to study the structure of DNA. In this technique, X-rays are diffracted as they passed through aligned fibers of purified DNA. The diffraction pattern can be used to deduce the threedimensional shape of molecules. James Watson learned from their research that DNA was helical in shape, and he deduced the width of the helix and the spacing of nitrogenous bases. The width of the helix suggested that it was made up of two strands, contrary to a three-stranded model that Linus Pauling had recently proposed. Watson and his colleague Francis Crick began to work on a model of DNA with two strands, the double helix. Using molecular models made of wire, they placed the sugarphosphate chains on the outside and the nitrogenous bases on the inside of the double helix. This arrangement put the relatively hydrophobic nitrogenous bases in the molecule's interior. The sugar-phosphate chains of each strand are like the side ropes of a rope ladder. Pairs of nitrogenous bases, one from each strand, form rungs. The ladder forms a twist every ten bases. The nitrogenous bases are paired in specific combinations: adenine with thymine and guanine with cytosine. Pairing like nucleotides did not fit the uniform diameter indicated by the X-ray data. A purine-purine pair is too wide, and a pyrimidine-pyrimidine pairing is too short. Only a pyrimidine-purine pairing produces the 2-nm diameter indicated by the X-ray data. In addition, Watson and Crick determined that chemical side groups of the nitrogenous bases would form hydrogen bonds, connecting the two strands. 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. This finding explained Chargaff's rules. The base-pairing rules dictate the combinations of nitrogenous bases that form the rungs of DNA. However, this does not restrict the sequence of nucleotides along each DNA strand. The linear sequence of the four bases can be varied in countless ways.
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Each gene has a unique order of nitrogenous bases. In April 1953, Watson and Crick published a succinct, one-page paper in Nature reporting their double helix model of DNA. Concept 16.2 Many proteins work together in DNA replication and repair The specific pairing of nitrogenous bases in DNA was the flash of inspiration that led Watson and Crick to the correct double helix. The possible mechanism for the next step, the accurate replication of DNA, was clear to Watson and Crick from their double helix model.
During DNA replication, base pairing enables existing DNA strands to serve as templates for new complementary strands.
In a second paper, Watson and Crick published their hypothesis for how DNA replicates. Essentially, because each strand is complementary to the other, each can form a template when separated. The order of bases on one strand can be used to add complementary bases and therefore duplicate the pairs of bases exactly. When a cell copies a DNA molecule, each strand serves as a template for ordering nucleotides into a new complementary strand. One at a time, nucleotides line up along the template strand according to the base-pairing rules. The nucleotides are linked to form new strands. Watson and Crick's model, semiconservative replication, predicts that when a double helix replicates, each of the daughter molecules will have one old strand and one newly made strand. Other competing models, the conservative model and the dispersive model, were also proposed. Experiments in the late 1950s by Matthew Meselson and Franklin Stahl supported the semiconservative model proposed by Watson and Crick over the other two models. In their experiments, they labeled the nucleotides of the old strands with a heavy isotope of nitrogen (15N), while any new nucleotides were indicated by a lighter isotope ( 14N). Replicated strands could be separated by density in a centrifuge. Each model--the semiconservative model, the conservative model, and the dispersive model--made specific predictions about the density of replicated DNA strands.
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The first replication in the 14N medium produced a band of hybrid (15N-14N) DNA, eliminating the conservative model. A second replication produced both light and hybrid DNA, eliminating the dispersive model and supporting the semiconservative model.
A large team of enzymes and other proteins carries out DNA replication. It takes E. coli 25 minutes to copy each of the 5 million base
pairs in its single chromosome and divide to two form identical daughter cells. A human cell can copy its 6 billion base pairs and divide into daughter cells in only a few hours. This process is remarkably accurate, with only one error per ten billion nucleotides. More than a dozen enzymes and other proteins participate in DNA replication. Much more is known about replication in bacteria than in eukaryotes. The process appears to be fundamentally similar for prokaryotes and eukaryotes. The replication of a DNA molecule begins at special sites, origins of replication. In bacteria, this is a specific sequence of nucleotides that is recognized by the replication enzymes. These enzymes separate the strands, forming a replication bubble. Replication proceeds in both directions until the entire molecule is copied. In eukaryotes, there may be hundreds or thousands of origin sites per chromosome. At the origin sites, the DNA strands separate, forming a replication bubble with replication forks at each end. The replication bubbles elongate as the DNA is replicated, and eventually fuse. DNA polymerases catalyze the elongation of new DNA at a replication fork. As nucleotides align with complementary bases along the template strand, they are added to the growing end of the new strand by the polymerase. The rate of elongation is about 500 nucleotides per second in bacteria and 50 per second in human cells. In E. coli, two different DNA polymerases are involved in replication: DNA polymerase III and DNA polymerase I. In eukaryotes, at least 11 different DNA polymerases have been identified so far.
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Each nucleotide that is added to a growing DNA strand is a nucleoside triphosphate. Each has a nitrogenous base, deoxyribose, and a triphosphate tail. ATP is a nucleoside triphosphate with ribose instead of deoxyribose. Like ATP, the triphosphate monomers used for DNA synthesis are chemically reactive, partly because their triphosphate tails have an unstable cluster of negative charge. As each nucleotide is added to the growing end of a DNA strand, the last two phosphate groups are hydrolyzed to form pyrophosphate. The exergonic hydrolysis of pyrophosphate to two inorganic phosphate molecules drives the polymerization of the nucleotide to the new strand. The strands in the double helix are antiparallel. The sugar-phosphate backbones run in opposite directions. 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. The 5' 3' direction of one strand runs counter to the 3' 5' direction of the other strand. DNA polymerases can only add nucleotides to the free 3' end of a growing DNA strand. A new DNA strand can only elongate in the 5' 3' direction. Along one template strand, DNA polymerase III can synthesize a complementary strand continuously by elongating the new DNA in the mandatory 5' 3' direction. The DNA strand made by this mechanism is called the leading strand. The other parental strand (5' 3' into the fork), the lagging strand, is copied away from the fork. Unlike the leading strand, which elongates continuously, the lagging stand is synthesized as a series of short segments called Okazaki fragments. Okazaki fragments are about 1,0002,000 nucleotides long in E. coli and 100200 nucleotides long in eukaryotes. Another enzyme, DNA ligase, eventually joins the sugarphosphate backbones of the Okazaki fragments to form a single DNA strand. DNA polymerases cannot initiate synthesis of a polynucleotide. They can only add nucleotides to the 3' end of an existing chain that is base-paired with the template strand. The initial nucleotide chain is called a primer.
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In the initiation of the replication of cellular DNA, the primer is a short stretch of RNA with an available 3' end. The primer is 510 nucleotides long in eukaryotes. Primase, an RNA polymerase, links ribonucleotides that are complementary to the DNA template into the primer. RNA polymerases can start an RNA chain from a single template strand. After formation of the primer, DNA pol III adds a deoxyribonucleotide to the 3' end of the RNA primer and continues adding DNA nucleotides to the growing DNA strand according to the base-pairing rules. Returning to the original problem at the replication fork, the leading strand requires the formation of only a single primer as the replication fork continues to separate. For synthesis of the lagging strand, each Okazaki fragment must be primed separately. Another DNA polymerase, DNA polymerase I, replaces the RNA nucleotides of the primers with DNA versions, adding them one by one onto the 3' end of the adjacent Okazaki fragment. The primers are converted to DNA before DNA ligase joins the fragments together. In addition to primase, DNA polymerases, and DNA ligases, several other proteins have prominent roles in DNA synthesis. Helicase untwists the double helix and separates the template DNA strands at the replication fork. This untwisting causes tighter twisting ahead of the replication fork, and topoisomerase helps relieve this strain. Single-strand binding proteins keep the unpaired template strands apart during replication. To summarize, at the replication fork, the leading strand is copied continuously into the fork from a single primer. The lagging strand is copied away from the fork in short segments, each requiring a new primer. It is conventional and convenient to think of the DNA polymerase molecules as moving along a stationary DNA template. In reality, the various proteins involved in DNA replication form a single large complex, a DNA replication machine. Many protein-protein interactions facilitate the efficiency of this machine. For example, helicase works much more rapidly when it is in contact with primase. The DNA replication machine is probably stationary during the replication process.
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In eukaryotic cells, multiple copies of the machine may anchor to the nuclear matrix, a framework of fibers extending through the interior of the nucleus. The DNA polymerase molecules reel in the parental DNA and extrude newly made daughter DNA molecules.
Enzymes proofread DNA during its replication and repair damage in existing DNA.
Mistakes during the initial pairing of template nucleotides and complementary nucleotides occur at a rate of one error per 100,000 base pairs. DNA polymerase proofreads each new nucleotide against the template nucleotide as soon as it is added. If there is an incorrect pairing, the enzyme removes the wrong nucleotide and then resumes synthesis. The final error rate is only one per ten billion nucleotides. DNA molecules are constantly subject to potentially harmful chemical and physical agents. Reactive chemicals, radioactive emissions, X-rays, and ultraviolet light can change nucleotides in ways that can affect encoded genetic information. DNA bases may undergo spontaneous chemical changes under normal cellular conditions. Mismatched nucleotides that are missed by DNA polymerase or mutations that occur after DNA synthesis is completed can often be repaired. Each cell continually monitors and repairs its genetic material, with 100 repair enzymes known in E. coli and more than 130 repair enzymes identified in humans. In mismatch repair, special enzymes fix incorrectly paired nucleotides. A hereditary defect in one of these enzymes is associated with a form of colon cancer. In nucleotide excision repair, a nuclease cuts out a segment of a damaged strand. DNA polymerase and ligase fill in the gap. The importance of the proper functioning of repair enzymes is clear from the inherited disorder xeroderma pigmentosum. These individuals are hypersensitive to sunlight. Ultraviolet light can produce thymine dimers between adjacent thymine nucleotides. This buckles the DNA double helix and interferes with DNA replication. In individuals with this disorder, mutations in their skin cells are left uncorrected and cause skin cancer.
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The ends of DNA molecules are replicated by a special mechanism.
Limitations of DNA polymerase create problems for the linear DNA of eukaryotic chromosomes. The usual replication machinery provides no way to complete the 5' ends of daughter DNA strands. Repeated rounds of replication produce shorter and shorter DNA molecules. Prokaryotes do not have this problem because they have circular DNA molecules without ends. The ends of eukaryotic chromosomal DNA molecules, the telomeres, have special nucleotide sequences. Telomeres do not contain genes. Instead, the DNA typically consists of multiple repetitions of one short nucleotide sequence. In human telomeres, this sequence is typically TTAGGG, repeated between 100 and 1,000 times. Telomeres protect genes from being eroded through multiple rounds of DNA replication. Telomeric DNA tends to be shorter in dividing somatic cells of older individuals and in cultured cells that have divided many times. It is possible that the shortening of telomeres is somehow connected with the aging process of certain tissues and perhaps to aging in general. Telomeric DNA and specific proteins associated with it also prevents the staggered ends of the daughter molecule from activating the cell's system for monitoring DNA damage. Eukaryotic cells have evolved a mechanism to restore shortened telomeres in germ cells, which give rise to gametes. If the chromosomes of germ cells became shorter with every cell cycle, essential genes would eventually be lost. An enzyme called telomerase catalyzes the lengthening of telomeres in eukaryotic germ cells, restoring their original length. Telomerase uses a short molecule of RNA as a template to extend the 3' end of the telomere. There is now room for primase and DNA polymerase to extend the 5' end. It does not repair the 3'-end overhang, but it does lengthen the telomere. Telomerase is not present in most cells of multicellular organisms. Therefore, the DNA of dividing somatic cells and cultured cells tends to become shorter.
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Telomere length may be a limiting factor in the life span of certain tissues and of the organism. Normal shortening of telomeres may protect organisms from cancer by limiting the number of divisions that somatic cells can undergo. Cells from large tumors often have unusually short telomeres, because they have gone through many cell divisions. Active telomerase has been found in some cancerous somatic cells. This overcomes the progressive shortening that would eventually lead to self-destruction of the cancer. Immortal strains of cultured cells are capable of unlimited cell division. Telomerase may provide a useful target for cancer diagnosis and chemotherapy.
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