POB Ch. 13
The transformation of eukaryotic cells by DNA is often called transfection. This can be demonstrated using a genetic marker, a gene whose presence in the recipient cells confers an observable phenotype. When transforming both prokaryotes and eukaryotes, researchers often use a genetically determined selection marker, for example, antibiotic resistance or a nutritional requirement, which permits the growth of transformed cells but not of nontransformed cells.
A common marker in mammalian transfection experiments is a gene that confers resistance to the antibiotic neomycin. Transfection is achieved by various methods, including chemical treatments that allow the DNA to be taken up by the cells. Any cell can be transfected, even an egg cell. In the latter case, a whole new genetically transformed organism can result, known as a transgenic organism. Transformation in eukaryotes offers conclusive evidence that DNA is the genetic material.
DNA replication involves remarkable teamwork among various proteins that act on the DNA strands. Let's review the proteins involved in DNA replication in the order of their activity at the replication fork: DNA helicase unwinds the double helix and separates the two strands. Single-strand binding proteins bind to separated strands and prevent them from re-forming the double helix. DNA primase makes RNA primers. DNA polymerase links new nucleotides to form the new DNA strands and removes the primers. DNA ligase connects Okazaki fragments made by DNA polymerase to one another.
A single primer is needed for synthesis of the leading strand, but each Okazaki fragment requires its own primer to be synthesized by the primase. In bacteria, DNA polymerase III then synthesizes an Okazaki fragment by adding nucleotides to one primer until it reaches the primer of the previous fragment (Figure 13.15). At this point, DNA polymerase I removes the old primer and replaces it with DNA. Left behind is a tiny nick—the final phosphodiester linkage between the adjacent Okazaki fragments is missing. The enzyme DNA ligase catalyzes the formation of that bond, linking the fragments and making the lagging strand whole.
Experiments on bacteria and on viruses demonstrated that DNA is the genetic material. DNA from one genetic strain of bacteria was able to genetically transform another strain into the donor strain. Viral DNA was shown to be injected into a host cell and to genetically change that cell into a virus factory.
At the time of Griffith's experiments in the 1920s, what circumstantial evidence suggested to scientists that DNA might be the genetic material? DNA was located in the eukaryotic cell nucleus, where chromosomes carrying genes were located. The amount of DNA was the same in somatic cells of an organism, and halved in the products of meiosis, as expected by the genetic material. Different species had different amounts of DNA, just as they seemingly had different numbers of genes.
DNA POLYMERASES ARE LARGE DNA polymerases are much larger than their substrates (the dNTPs) and the template DNA, which is very thin. Molecular models of the enzyme-substrate-template complex from bacteria show that the enzyme is shaped like an open right hand with a palm, a thumb, and fingers (Figure 13.12). Within the "palm" is the active site of the enzyme, which brings together each dNTP substrate and the template. The "finger" regions have precise shapes that can recognize the different shapes of the four nucleotide bases. They bind to the bases by hydrogen bonding and rotate inward. Most cells contain more than one kind of DNA polymerase, but only one of them is responsible for chromosomal DNA replication. The others are involved in primer removal and DNA repair. Fifteen DNA polymerases have been identified in humans, whereas the bacterium E. coli has five DNA polymerases.
DNA REPLICATION BEGINS WITH A PRIMER As DNA polymerase elongates a polynucleotide strand by covalently linking new nucleotides to a preexisting strand. However, it cannot start this process without a short "starter" strand, called a primer. In most organisms this primer is a short single strand of RNA (Figure 13.11), but in some organisms it is DNA. The primer is complementary to the DNA template and is synthesized one nucleotide at a time by an enzyme called a primase. The DNA polymerase then adds nucleotides to the 3′ end of the primer and continues until the replication of that section of DNA has been completed. Then the RNA primer is degraded, DNA is added in its place, and the resulting DNA fragments are connected by the action of other enzymes. When DNA replication is complete, each new strand consists only of DNA.
Replication of a Chromosome A eukaryotic chromosome contains a long, linear molecule of double-stranded DNA. This DNA is tightly coiled within the chromosome, but unravels during interphase, when DNA replication occurs. A eukaryotic chromosome contains multiple origins of replication. At each origin of replication, DNA synthesis proceeds bidirectionally. Two replication forks move outward in opposite directions. The replicating DNA helices from each origin elongate and eventually join each other. The original chromosome has been replicated to form two identical daughter DNA molecules, also called sister chromatids.
DNA polymerases require a preexisting RNA or DNA strand, the primer, to initiate new DNA synthesis. These polymerases add deoxyribonucleotides only to the 3' end of a growing strand. Taking a closer look at the nucleotide polymerization reaction, the 3' end of the primer contains a free 3'-hydroxyl group. The 3' hydroxyl reacts with the 5' end of the next free nucleotide to be added. Free nucleotides continue to be added to the growing DNA strand by the same type of reaction. Overall, the new DNA strand grows in the 5'-to-3' direction.
ORIGINS OF REPLICATION The single circular chromosome of the bacterium E. coli has 4 × 106 base pairs (bp) of DNA. The 245 bp ori sequence is at a particular location on the chromosome. Once the pre-replication complex binds to it, the DNA is unwound and replication proceeds in both directions around the circle, forming two replication forks (Figure 13.10A). The replication rate in E. coli is approximately 1,000 bp per second, so it takes about 40 minutes to fully replicate the chromosome (with two replication forks). Rapidly dividing E. coli cells divide every 20 minutes. In these cells, new rounds of replication begin at the ori of each new chromosome before the first chromosome has fully replicated. In this way the cells can divide more frequently than the time needed to finish replicating the original chromosome.
DNA replication begins with the binding of a large protein complex (the pre-replication complex) to a specific site on the DNA molecule. This complex contains several different proteins, including the enzyme DNA polymerase, which catalyzes the addition of nucleotides as the new DNA chain grows. All chromosomes have at least one region called the origin of replication (ori), to which the pre-replication complex binds. Binding occurs when proteins in the complex recognize specific DNA sequences within the ori.
Suppose proteins, and not DNA, were the genetic material in bacteriophage T2. What would we expect to find in the host cells in the Hershey and Chase blender experiment? Please choose the correct answer from the following choices, and then select the submit answer button. Correct: Most of the labeled S Most of the labeled S
DNA replication progresses in the direction of the acting helicase. Challenge this Question Which statement about DNA replication pertains specifically to synthesis of the leading strand? Please choose the correct answer from the following choices, and then select the submit answer button.
Test tube synthesis of DNA needed the following substances: 1. The deoxyribonucleoside triphosphates dATP, dCTP, dGTP, and dTTP. These are the monomers from which the DNA polymers are formed. 2. DNA molecules of a particular sequence that serve as templates to direct the sequence of nucleotides in the new molecules. 3. A DNA polymerase enzyme to catalyze the polymerization reaction. 4. Salts and a pH buffer, to create an appropriate chemical environment for the DNA polymerase.
The fact that DNA could be synthesized in a test tube confirmed that a DNA molecule contains the information needed for its own replication. The next challenge was to determine which of three possible replication patterns occurs during DNA replication: Semiconservative replication, in which each parent strand serves as a template for a new strand, and the two new DNA molecules each have one old and one new strand (Figure 13.8A) Conservative replication, in which the original double helix serves as a template for, but does not contribute to, a new double helix (Figure 13.8B) Dispersive replication, in which fragments of the original DNA molecule serve as templates for assembling two new molecules, each containing old and new parts, perhaps at random (Figure 13.8C)
To satisfy Chargaff's rule (A = T and G = C), Watson and Crick's model always paired a purine on one strand with a pyrimidine on the opposite strand. These base pairs (A-T and G-C) have the same width down the double helix, a uniformity that was also confirmed by X-ray diffraction. Crick and Watson built their tin model of DNA in late February 1953. This structure explained the known chemical properties of DNA, and it opened the door to understanding its biological functions.
WATSON AND CRICK To be consistent with Franklin's X-ray diffraction images, Watson and Crick's model had the nucleotide bases on the interior of the two strands, with a sugar-phosphate "backbone" on the outside. In addition, the two DNA strands ran in opposite directions, that is, they were antiparallel. The two strands would not fit together otherwise.
Watson and Crick used X-ray diffraction data in conjunction with chemical evidence to build the double helix model for DNA. The genetic material performs four important functions and is well suited to its biological functions.
What attributes of bacteriophage T2 were key to the Hershey-Chase experiments demonstrating that DNA, rather than protein, is the genetic material? Bacteriophage T2 has only two types of molecules, DNA and protein. So labeling one or the other could indicate which got into a host cell to cause genetic changes.
One way to quickly identify an organism in the field is to use PCR: If there is amplification of a particular sequence, then the organism is identified. What is the basis for this test? If a primer has a species-specific sequence—that is, a sequence of nucleotides complementary to a sequence unique to a certain organism—PCR will amplify a target DNA extracted from a field sample that has the unique sequence. This amplification will show that the organism with that sequence is present
What is the role of primers in PCR? Primers in PCR bind to a short region of DNA and allow elongation of that strand of DNA when DNA polymerase is added along with nucleotide.
An enzyme called DNA helicase uses energy from ATP hydrolysis to unwind and separate the strands, and single-strand binding proteins bind to the unwound strands to keep them from reassociating into a double helix. This process makes each of the two template strands available for complementary base pairing.
What structural property of DNA gave Watson and Crick the idea that DNA replication was likely to proceed via a semiconservative mechanism? B. The complementary nature of base pairing between the two strands
How did the experiments of Avery and his colleagues rule out protein as the genetic material?
When they pretreated the extract of the donor strain with enzymes that hydrolyzed proteins, transformation still occurred. So proteins in the donor extract were not responsible for genetic transformation.
Errors in DNA can occur during replication and as the result of chemical changes in bases. It gets worse: errors in base pairs can arise spontaneously as well. Because the bases themselves are chemically instable, outside agents like radiation can damage them, causing *mutations that prevent them from pairing properly.
Why are there special sequences at the ends of chromosomes, and how are they replicated? The special sequences at the ends of chromosomes are called telomeres, and they can be replicated if necessary. After DNA replication, the primer regions at the 3′ ends of the long DNA in chromosomes are removed. This shortens the DNA and makes it unstable. In some cells, such as gamete-producing cells and cancer cells, the telomeric sequences are recognized by an enzyme complex called telomerase, which catalyzes the replication of any lost telomeric sequences and keeps the DNA at its original length.
Assuming you have four copies of your desired template DNA to start, and you run a 25-cycle PCR, how many copies of DNA will you have at the end of the PCR (under optimal conditions)? A. 4 × 225
With PCR, it is possible to amplify a single piece of DNA and generate millions of copies of the original DNA molecule. By using PCR in diagnostic tests, researchers can determine whether a particular pathogen is present in a sample. The test is set up so that the reaction mixture contains a pair of oligonucleotide primers that match up with a region of a pathogen's DNA. If the pathogen is present in the sample, a fragment of its DNA will be amplified by the PCR technique, producing a positive result. Primers can be created to identify any pathogen with a known DNA sequence. Because PCR requires very little starting material, a small test sample is all that is required to set up many reactions, each using a primer pair corresponding to a different pathogen.
When an enzyme is able to catalyze multiple reactions each time it binds to its substrate, thereby creating multiple products, it is said to be _______. Processive
a factor from S strain was modifying the R strain bacteria. Griffith's experiments, which showed the transformation of R strain pneumococcus bacteria to S strain pneumococcus bacteria in the presence of heat-killed S strain bacteria, provided evidence that?
The genetic material is susceptible to mutations (permanent changes) in the information it encodes. Challenge this Question What structural feature of DNA can disrupt the specificity of a protein-DNA interaction? Please choose the correct answer from the following choices, and then select the submit answer button.
the DNA polymerase enzyme requires the 3′ (-OH) end of an existing strand to catalyze the addition of deoxyribonucleotides. Challenge this Question RNA primers are necessary in DNA synthesis because Please choose the correct answer from the following choices, and then select the submit answer button.
✔ Correct! Okazaki fragments are the newly formed DNA making up the lagging strand in DNA replication. While the leading strand grows continuously "forward," the lagging strand grows in shorter, "backward" stretches with gaps between them.
✔ Correct! In DNA replication, the lagging strand is the daughter strand that is synthesized in discontinuous stretches. The lagging strand is oriented so that as the fork opens up, its exposed 3′ end gets farther and farther away from the fork, and an unreplicated gap is formed.
✔ Correct! DNA polymerase is any of a group of enzymes that catalyze the formation of DNA strands from a DNA template. DNA polymerases add nucleotides to the growing chain.
✔ Correct! In DNA replication, the leading strand is the daughter strand that is synthesized continuously.
Suppose proteins, and not DNA, were the genetic material in bacteriophage T2. What would we expect to find in the host cells in the Hershey and Chase blender experiment? Most of the labeled S
Pure protein from S cells added to R cells Suppose the actual transforming substance had been found to be protein and not DNA. Which scenario would be expected to produce a transformation that would cause mice to become sick?
The nucleotides that make up DNA are deoxyribonucleoside monophosphates because they each contain deoxyribose and one phosphate group (see Figure 4.1). The four free monomers that are brought together to form DNA are the deoxyribonucleoside triphosphates dATP, dTTP, dCTP, and dGTP, collectively referred to as dNTPs. They are called triphosphates because each has three phosphate groups. The three phosphate groups are attached to the 5′ carbon on the deoxyribose sugar. Note that during DNA replication nucleotides are added to the growing new strand at the 3′ end—the end at which the DNA strand has a free hydroxyl (—OH) group on the 3′ carbon of its terminal deoxyribose. In the formation of the phosphodiester linkage (a condensation reaction), two of the phosphate groups on an incoming dNTP are removed [as pyrophosphate (PPi)], and the remaining phosphate is bonded to the 3′ carbon on the terminal deoxyribose (see Figure 4.2). Just as energy is released when ATP is hydrolyzed to AMP (with subsequent hydrolysis of PPi to two phosphates), energy is released by the hydrolysis of the dNTP, and this energy is used to drive the condensation reaction.
Semiconservative DNA replication in the cell involves several different enzymes and other proteins. It takes place in two general steps: The DNA double helix is unwound to separate the two template strands and make them available for new base pairing. As new nucleotides form complementary base pairs with template DNA, they are covalently linked together by phosphodiester bonds, forming a polymer whose base sequence is complementary to the bases in the template strand.
ANTIPARALLEL STRANDS The backbone of each DNA strand contains repeating units of the five-carbon monosaccharide deoxyribose: The number followed by a prime (′) designates the position of a carbon atom in this sugar molecule. In the sugar-phosphate backbone of DNA, the phosphate groups are connected to the 3′ carbon of one deoxyribose molecule and the 5′ carbon of the next, linking successive sugars together. Thus the two ends of a polynucleotide chain differ. At one end of a chain is a free (not connected to another nucleotide) 5′ phosphate group (—OPO3-); this is called the 5′ end. At the other end is a free 3′ hydroxyl group (—OH); this is called the 3′ end. In a DNA double helix, the 5′ end of one strand is paired with the 3′ end of the other strand, and vice versa. In other words, if you drew an arrow for each strand running from 5′ to 3′, the arrows would point in opposite directions (see also Figure 4.4A).
THE HELIX The sugar-phosphate backbones of the polynucleotide chains form a coil around the outside of the helix, and the nitrogenous bases point toward the center. The chains are held together by two chemical forces: 1. Hydrogen bonding between specifically paired bases. Consistent with Chargaff's rule, adenine (A) pairs with thymine (T) by forming two hydrogen bonds, and guanine (G) pairs with cytosine (C) by forming three hydrogen bonds: **Every base pair consists of one purine (A or G) and one pyrimidine (T or C). This pattern is known as complementary base pairing. 2. van der Waals forces between adjacent bases on the same strand. When the base rings come near one another, they tend to stack like poker chips because of these weak attractions.
One newly replicating strand (the leading strand) is oriented so that it can grow continuously at its 3′ end as the fork opens up. The other new strand (the lagging strand) is oriented so that as the fork opens up, its exposed 3′ end gets farther and farther away from the fork, and an unreplicated gap is formed. This gap would get bigger and bigger if there were not a special mechanism to overcome this problem.
The DNA at the replication fork—the site where DNA unwinds to expose the bases so that they can act as templates—opens up like a zipper in one direction.
Name five proteins needed for DNA replication. What are their roles? The five proteins involved in DNA replication are DNA helicase (unwinds the double helix), single-strand binding proteins (stabilize and keep apart the two strands in unwound regions), primase (binds to DNA to make a short primer), DNA polymerase (adds nucleotides to a growing chain), and DNA ligase (seals up nicks in DNA, due to its lagging strand replication in short pieces).
The bacteria were prelabeled with both strands of DNA with heavy 15N. After one round of replication in light 14N, three models for DNA replication had different predictions: Conservative: There would be original DNA (all heavy) and new DNA (all light) in equal amounts. This did not occur. Dispersive: There would be light, heavy, and intermediate DNA in no fixed proportions. This did not occur. Semiconservative: There would be only intermediate DNA, with one light and one heavy strand. This did occur.
DNA IS THREADED THROUGH A REPLICATION COMPLEX So far, you are probably envisioning DNA replication as a locomotive (the replication complex) moving along a railroad track (the DNA). But this is not so. Commonly in eukaryotes, the replication complexes seem to be stationary, attached at specific positions in the nucleus. It is the DNA that moves, essentially sliding into the replication complex as one double-stranded molecule and emerging as two double-stranded molecules.
DNA replication would not proceed as rapidly as it does if it went through such a cycle for each nucleotide. Instead, DNA polymerases are processive—that is, they catalyze the formation of many phosphodiester linkages each time they bind to a DNA molecule: Substrates bind to enzyme → many products are formed → enzyme is released → cycle repeats The DNA polymerase-DNA complex is stabilized by a sliding DNA clamp, which has multiple identical subunits assembled into a doughnut shape (Figure 13.16). The doughnut's "hole" is just large enough to encircle the DNA double helix, along with a thin layer of water molecules for lubrication. The clamp binds to the DNA polymerase-DNA complex, keeping the enzyme and the DNA associated tightly with each other. If the clamp is absent, DNA polymerase dissociates from DNA after forming 20 to 100 phosphodiester linkages. With the clamp, it can polymerize up to 50,000 nucleotides before it detaches.
How does the two-stranded structure of DNA relate to its functions? The double-stranded structure is essential in the replication of DNA, as the opposite strands can each act as a template for a new strand, so that two new identical strands are made. This is key in the replication of the genetic material when cells divide. The two strands can unravel at places, exposing the bases in the inside for gene expression. This is important because genes must be expressed for the phenotype.
Describe the evidence that Watson and Crick used to come up with the double helix model for DNA. X-ray diffraction indicated that DNA is double-stranded and twisted into a helix. There were indications that the bases were inside the helix, and the sugars and phosphates on the outside. Base composition data from many organisms showed that the percentages of the purine A = those of the pyrimidine T, and that the percentages of the purine G = those of the pyrimidine C. This suggested that A might be opposite T on the inside of the double helix, and G opposite C. When Watson and Crick built molecular models with the atoms and bonds of polynucleotide strands, the base pairing was confirmed, as the A-T and G-C pairs fit nicely together.
DNA replication is not perfect. In addition, DNA may be altered or damaged by environmental factors. Repair mechanisms detect and repair mismatched or damaged DNA.
Fortunately, our cells correct DNA replication errors and repair damaged nucleotides. Cells have at least three DNA repair mechanisms at their disposal: A proofreading mechanism corrects errors in replication as DNA polymerase makes them. A mismatch repair mechanism scans DNA immediately after it has been replicated and corrects any base-pairing mismatches. An excision repair mechanism removes abnormal bases that have formed because of chemical damage and replaces them with functional bases.
In DNA, where do the following chemical forces occur: hydrogen bonds, covalent bonds, and van der Waals forces? Hydrogen bonds occur between the bases on opposite strands, within base pairs. Covalent bonds occur between the atoms that make up nucleotides and between the nucleotides in a DNA strand. Van der Waals forces occur between the flat bases that stack on top of each other within the double helix, stabilizing them in the stacking.
Four features summarize the molecular architecture of the DNA molecule (Figure 13.6B): DNA is a double-stranded helix, with a sugar-phosphate backbone on the outside and base pairs lined up on the inside. DNA is usually a right-handed helix. If you curl the fingers of your right hand and point your thumb upward, the curve of the helix follows the direction of your fingers, and it winds upward in the direction of your thumb. (See Figure 3.8, of right- versus left-handed helices.) DNA is antiparallel (the two strands run in opposite directions). DNA has major and minor grooves in which the outer edges of the nitrogenous bases are exposed.
The polymerase chain reaction (PCR) technique essentially automates this replication process by copying a short region of DNA many times in a test tube. The PCR reaction mixture contains: A sample of double-stranded DNA from a biological sample, to act as the template Two short, artificially synthesized primers that are complementary to the ends of the sequence to be amplified The four dNTPs (dATP, dTTP, dCTP, and dGTP) A DNA polymerase that can tolerate high temperatures without becoming degraded Salts and a buffer to maintain a near-neutral pH
How do PCR primers relate to DNA replication primers? Both PCR primers in the test tube and DNA replication primers in the cell act to begin DNA replication. However, PCR primers can be complementary to any DNA strand, whereas DNA replication primers bind only to the origin of replication. The first step involves heating the reaction mixture to near boiling point, to separate (denature) the two strands of the DNA template. The reaction is then cooled to allow the primers to bind (or anneal) to the template strands. Next, the reaction is warmed to an optimum temperature for the DNA polymerase to catalyze the production of the complementary new strands.
The Meselson-Stahl experiment provided evidence to support the semiconservative model of DNA replication. Semiconservative DNA replication requires deoxyribonucleoside triphosphates, DNA polymerase, and a DNA template. DNA replication proceeds bidirectionally from an origin of replication (ori); E. coli has a single ori, and eukaryotic chromosomes have multiple ones. The first replication event at the ori uses DNA helicases and single-stranded binding proteins.
How do specific interactions between proteins and DNA occur? The bases in DNA expose chemical groupings that can interact with groups on proteins. These include polar groups (e.g., C=O) on the bases that can attract oppositely polar groups (e.g., NH2) on proteins, as well as form hydrogen bonds with groups on proteins.
Ligases are enzymes that link two DNA fragments together. Suppose a bacterium develops a mutation in the ligase gene needed for DNA replication. This mutation results in an inactive form of the enzyme. What outcome can you expect for this bacterium and why? A. The bacterium will not be able to replicate itself because only one DNA strand will result from the replication process instead of two
How does synthesis of the leading strand differ from synthesis of the lagging strand during DNA replication? C. More primers are needed per lagging strand synthesized than are needed per leading strand synthesized.
To prove that DNA is the transforming substance, Avery, MacLeod, and McCarty demonstrated that DNA could transform nonvirulent strains of pneumococcus. The definitive evidence for transformation was? The transformed R bacteria passed on the virulence trait to daughter cells.
If one strand of DNA has a C attached to a sugar with a free 5′ phosphate group, the other strand will have a _______ attached to a sugar with a free _______ group. G; 3' hydroxyl group
telomeres: (tee´ lo merz) [Gk. telos: end + meros: units, segments] Repeated DNA sequences at the ends of eukaryotic chromosomes.
In many eukaryotes, there are repetitive sequences at the ends of chromosomes called telomeres. In humans, the telomere sequence is TTAGGG-3′, and it is repeated about 2,500 times at each chromosome end. These repeats bind special proteins that prevent the DNA repair system from recognizing the chromosome ends as breaks. In addition, the repeats may form loops that have a similar protective role. But there is another problem with chromosome ends. As you have seen, replication of the lagging strand occurs by the addition of Okazaki fragments to RNA primers. When the terminal RNA primer is removed, no DNA can be synthesized to replace it because there is no 3′ end to extend. In most cells, the short piece of single-stranded DNA at each end of the chromosome is removed. Thus the chromosome becomes slightly shorter with each cell division
A researcher would like to radioactively label DNA for an experiment. Which type of molecule could not serve as a target for radioactive labeling? Sulfur
In the Hershey and Chase experiment an isotope of _______ was used to label protein. Sulfur
transfection: Insertion of recombinant DNA into animal cells.
In the Hershey-Chase experiment, which radioisotopes were used to label which biomolecules that make up a virus? 32P in nucleic acids; 35S in proteins
PHYSICAL EVIDENCE FROM X-RAY DIFFRACTION The positions of atoms in a crystallized substance can be inferred from the diffraction pattern of X rays passing through the substance. In the early 1950s the New Zealand-born biophysicist Maurice Wilkins discovered a way to make highly ordered fibers of DNA that were suitable for X-ray diffraction studies. His samples were analyzed by Rosalind Franklin of Kings College, London (Figure 13.5). Franklin's data suggested that DNA was a double (two-stranded) helix with ten nucleotides in each full turn, and that each full turn was 3.4 nanometers (nm) in length. The molecule's diameter of 2 nm suggested that the sugar-phosphate backbone of each DNA strand must be on the outside of the helix.
In the early 1950s, biochemist Erwin Chargaff and his colleagues at Columbia University reported that DNA from many different species—and from different sources within a single organism—exhibits certain regularities. This led to the following rule: In any DNA sample, the amount of adenine equals the amount of thymine (A = T), and the amount of guanine equals the amount of cytosine (G = C). As a result, the total abundance of purines (A + G) equals the total abundance of pyrimidines (T + C)
If the proportion of C in a double-stranded DNA molecule is x, what is the proportion of T? 1/2 - x
Okazaki fragments are synthesized as part of the lagging strand. Which feature of DNA replication is a consequence of semiconservative replication?
The polymerase chain reaction was developed for the purpose of rapidly making multiple copies of segments of DNA.
One form of colon cancer is caused by a mutation in a gene encoding a protein involved in mismatch repair. What would be the consequences of such a mutation in cancer formation? Recall cell cycle control genes involved in cancer formation. If the colon cancer cell had a mutation in a tumor suppressor gene, it might not be repaired. This would lead to unregulated cell division.
The genetic material performs four important functions, and the DNA structure proposed by Watson and Crick was elegantly suited to three of them. 1. The genetic material stores an organism's genetic information. With its millions of nucleotides, the base sequence of a DNA molecule can encode and store an enormous amount of information. Variations in DNA sequences can account for species and individual differences. DNA fits this role nicely. 2. The genetic material is susceptible to mutations (permanent changes) in the information it encodes. For DNA, mutations might be simple changes in the linear sequence of base pairs. 3. The genetic material is precisely replicated in the cell division cycle. Replication could be accomplished by complementary base pairing, A with T and G with C. In the original publication of their findings in 1953, Watson and Crick coyly pointed out, "It has not escaped our notice that the specific pairing we have postulated immediately suggests a possible copying mechanism for the genetic material." 4. The genetic material (the coded information in DNA) is expressed as the phenotype. This function is not obvious in the structure of DNA. However, as we will see in the next chapter, the nucleotide sequence of DNA is copied into RNA, which uses the coded information to specify a linear sequence of amino acids—a protein. The folded forms of proteins determine many of the phenotypes of an organism.
BASE EXPOSURE IN THE GROOVES Look back at Figure 13.6B and note the major and minor grooves in the helix. These grooves exist because the backbones of the two strands are closer together on one side of the double helix (forming the minor groove) than on the other side (forming the major groove). The exposed outer edges of the base pairs are accessible for additional hydrogen bonding. Note that the arrangements of unpaired atoms and groups differ in A-T and G-C pairs. Thus, the surfaces of the A-T and C-G base pairs are chemically distinct, allowing other molecules such as proteins to recognize specific base pair sequences and bind to them, which is crucial for DNA function. The binding of proteins to specific base pair sequences is the key to protein-DNA interactions, which are necessary for the replication and expression of the genetic information in DNA.
IN IDEA THAT DNA IS THE GENETIC MATERIAL At this time a new dye was developed that could bind specifically to DNA and that stained cell nuclei red in direct proportion to the amount of DNA present in the cell. This technique provided circumstantial evidence that DNA was the genetic material: DNA was in the right place. DNA was confirmed to be an important component of the nucleus and the chromosomes, which were known to carry genes. DNA was present in the right amounts. The amount of DNA in somatic cells (body cells not specialized for reproduction) was twice that in reproductive cells (eggs or sperm)—as might be expected for diploid and haploid cells, respectively. DNA varied among species. When cells from different species were stained with the dye and their color intensity measured, each species appeared to have its own specific amount of nuclear DNA.
Cell staining techniques provided the first pieces of evidence that DNA was the genetic material. Experiments showed that one strain of bacteria could be genetically transformed to another strain and that DNA was the transforming agent. Experiments using radiolabeled protein and DNA identified DNA as the material injected by a virus during its infection of a host cell.
telomerase: An enzyme that catalyzes the addition of telomeric sequences lost from chromosomes during DNA replication.
Continuously dividing cells, such as bone marrow stem cells and gamete-producing cells, have a special mechanism for maintaining their telomeric DNA. An enzyme called telomerase catalyzes the addition of any lost telomeric sequences in these cells (see Figure 13.18). Telomerase contains an RNA sequence that acts as a template for the telomeric DNA repeat sequence.
Hershey and Chase set out to determine what part of the virus—DNA or protein—enters the host cell to bring about this genetic change. To trace the two components of the virus over its life cycle, the scientists labeled each component with a specific radioisotope: Proteins were labeled with radioactive sulfur. Proteins contain some sulfur (in the amino acids cysteine and methionine), but DNA does not. Sulfur has a radioactive isotope, 35S. Hershey and Chase grew bacteriophage T2 in a bacterial culture in the presence of 35S, so the proteins of the resulting viruses were labeled with (contained) the radioisotope. DNA was labeled with radioactive phosphorus. DNA contains a lot of phosphorus (in the deoxyribose-phosphate backbone—see Figure 4.4), whereas proteins contain little or none. Phosphorus also has a radioisotope, 32P. The researchers grew another batch of T2 in a bacterial culture in the presence of 32P, thus labeling the viral DNA with 32P.
Oswald Avery and his colleagues at what is now The Rockefeller University identified the substance causing bacterial transformation in two ways: Eliminating other possibilities. Cell-free extracts containing the transforming substance were treated with enzymes that destroyed candidates for the genetic material, such as proteins, RNA, and DNA. When the treated samples were tested, the ones treated with RNase and protease (which destroy RNA and proteins, respectively) were still able to transform R-type bacteria into the S-type. But the transforming activity was lost in the extract treated with DNase (which destroys DNA) (Figure 13.2). Positive experiment. The researchers isolated virtually pure DNA from a cell-free extract containing the transforming substance. The DNA alone caused bacterial transformation.
Not long after this experiment, other scientists investigated DNA replication in the presence of cisplatin (described in the opening story of this chapter). Repeating the Meselson-Stahl experiment with cisplatin added to the growth media, they found no change in the density of DNA after several generations—that is, only one DNA band formed in the tubes subjected to centrifugation. From this the researchers deduced that in the presence of cisplatin the two strands of DNA must not be separating during replication. We will discuss more specifically how cisplatin prevents DNA strand separation at the end of this chapter. First, let's consider the chemistry involved in DNA replication. The Meselson-Stahl experiment showed that DNA replicates by a semiconservative mechanism. The double helix separates so that each old strand serves as a template for a new strand. Two new double helices result, each containing one new strand and one old strand.
The key to the experiment was the use of a "heavy" isotope of nitrogen. Heavy nitrogen (15N) is a rare, nonradioactive isotope that makes molecules containing it denser than chemically identical molecules containing the common isotope 14N. Two cultures of the bacterium E. coli were grown for many generations, one in a medium containing 15N and the other in a medium containing 14N. When DNA extracts from the two cultures were combined and centrifuged in a solution of cesium chloride, which forms a density gradient under centrifugation, two separate bands of DNA formed in the centrifugation tube. The DNA from the 15N culture was heavier than the DNA from the 14N culture, so it formed a band at a different position in the density gradient. Figure A in the work with the data portion of Investigating Life: The Meselson-Stahl Experiment shows a photo of the two bands. Next, Meselson and Stahl grew another E. coli culture in 15N medium, then transferred the bacteria to normal 14N medium and allowed them to continue growing. The cells replicated their DNA and divided every 20 minutes. Meselson and Stahl collected some of the bacteria at time intervals and extracted DNA from the samples. You can follow their results for the first two generations in Investigating Life: The Meselson-Stahl Experiment. The results can be explained only by the semiconservative model of DNA replication. The crucial observations demonstrating this model were that all the DNA at the end of the first generation was of intermediate density, while at the end of the second generation there were two discrete bands: one of intermediate and one of light DNA. If the conservative model had been true, there would have been no intermediate density DNA. If the dispersive model were correct, then the DNA would all have been intermediate for the first few generations, with the single intermediate band becoming progressively lighter.
Xeroderma pigmentosum (XP) is a hereditary disease in which there is a defect in the repair system that repairs DNA after it is damaged by ultraviolet rays. The symptoms of XP are likely due to an _______ repair system. Under active excision
The nucleotides that make up a DNA molecule each contain _______ phosphate group(s). Before they are added to the DNA, the precursors to nucleotides each contain _______ phosphate group(s). One; three
S-type cells have a protective smooth capsule, and R-type cells do not. In Griffith's experiment, why were S-type cells virulent and R-type cells not virulent?
The proofreading DNA repair system differs from other types of DNA repair in that it? Occurs as DNA is being replicated
