11: DNA and Its Role in Heredity

DNA: The Genetic Material

· During the first half of the twentieth century, the structure that carried hereditary information from generation to generation was generally assumed to be a protein.

· The diversity and specificity of proteins seemed appropriate for genetic material.

· DNA seemed too simple to carry complex information.

· Circumstantial evidence, however, pointed to DNA as the genetic material.

· DNA was found in the nucleus, which was already known to carry genes.

· A dye that bound to DNA showed that the amount of DNA in somatic cells was twice that in eggs or sperm, as would be expected from Mendel’s discoveries.

DNA from one type of bacterium genetically transforms another type

· In the 1920s, the English physician Frederick Griffith made a landmark discovery about heredity while looking for a vaccine against Streptococcus pneumoniae, one of the bacteria that cause pneumonia in humans.

· Griffith worked with two different strains of the bacterium. (See Figure 11.1.)

· The S strain produced shiny, smooth colonies when grown in the laboratory.

· The R strain produced colonies that looked rough.

· The S strain was virulent (mice injected with the S strain died within a day); a capsule around the S strain bacteria protected them from the host’s immune system.

· The R strain lacked this capsule and was nonvirulent.

· Griffith heated some S strain bacteria to kill them, then injected the bacteria into mice.

· The heat-killed bacteria did not kill the mice.

· A mixture of heat-killed S strain bacteria and living R strain bacteria, however, did kill the mice.

· Griffith found living S strain bacteria in the hearts of the mice killed in this way.

· He concluded that some of the living R strain bacteria had been transformed by the presence of the heat-killed S strain bacteria.

· Further tests demonstrated that some substance from the dead S strain bacteria could cause a heritable change in the R strain bacteria.

· Some scientists concluded that this “transforming principle” carried heritable information and thus was the genetic material.

The transforming principle is DNA

· Oswald T. Avery and colleagues spent several years identifying the transforming principle by a process of elimination.

· They treated the extract from heat-killed S strain bacteria in various ways to destroy different types of substances but retain others.

· Invariably, when DNA was destroyed, the transforming activity was lost, but when DNA was left intact, the transforming activity survived.

· This work, published in 1944, was not immediately appreciated.

· Little was known about bacterial genetics, and it was not yet obvious that bacteria even had genes.

Viral replication experiments confirm that DNA is the genetic material

· In 1952, Alfred D. Hershey and Martha Chase performed experiments confirming that DNA is the genetic material.

· The T2 bacteriophage, a virus that attacks E. coli, consists almost entirely of a DNA core packed in a protein coat. (See Figure 11.2. Chapter 13 of the Instructor’s Resource CD-ROM includes micrographs of similar bacteriophages.)

· When a T2 bacteriophage attacks a bacterium, part but not all of the virus enters the bacterial cell.

· The Hershey–Chase experiment determined which part of the virus entered the bacterium. (See Figure 11.3.)

· Some viruses were labeled with radioactive sulfur. Sulfur is present in proteins but not in DNA.

· Other viruses were labeled with radioactive phosphorus. Phosphorus is present in DNA but absent from most proteins.

· In separate experiments, viruses with labeled sulfur and labeled phosphorus were combined with bacteria.

· Blending the resulting bacteria removed the viral material that had not entered the bacteria.

· Centrifuging revealed that the labeled sulfur (and thus the viral protein) had separated from the bacteria, but the labeled phosphorus (and thus the viral DNA) remained with the bacteria.

· Experiments on later generations of bacteria confirmed that the labeled phosphorus remained with subsequent generations while the labeled sulfur was quickly lost.

· By this time, it was accepted that bacteria had genes, so the Hershey–Chase experiment convinced most scientists that DNA was the carrier of hereditary information.

The Structure of DNA

· Scientists set out to determine the structure of DNA hoping to find the answers to two questions:

· How is DNA replicated between nuclear divisions?

· How does DNA cause the synthesis of specific proteins?

X-ray crystallography provided clues to DNA structure

· The positions of atoms in a crystalline substance can be inferred from the pattern of diffraction of X-rays passed through it. (See Figure 11.4. The Instructor’s Resource CD-ROM includes an X-ray diffraction pattern from DNA.)

· In the early 1950s, many skilled X-ray crystallographers tried but failed to glean information from X-ray diffraction patterns of DNA.

· The English chemist Rosalind Franklin, building on previous work by Maurice Wilkins, was able to provide key information about the structure of DNA based on X-ray crystallography.

The chemical composition of DNA was known

· By the 1950s it was known that DNA was a polymer of nucleotides. (See Figures 3.16 and 3.17.)

· The four nucleotides that make up DNA differ only in their nitrogenous bases.

· There are two purines (adenine and guanine) and two pyrimidines (cytosine and thymine).

· In 1950, Erwin Chargaff noted that in DNA from all species tested, the amount of adenine equals the amount of thymine, and the amount of guanine equals the amount of cytosine. (See Figure 11.5.)

· In other words, the total abundance of purines equals the total abundance of pyrimidines, even though the actual proportions of each base vary in different species.

Watson and Crick described the double helix

· The English physicist Francis Crick and the American geneticist James D. Watson used the technique of model building to establish the general structure of DNA.

· The results of X-ray crystallography convinced them that the DNA molecule was helical.

· X-ray crystallography also provided the values of certain distances within the helix.

· Density measurements and earlier models pointed to a structure with two polynucleotide chains running antiparallel to each other.

· Although there have been modifications, the principle features of the model they built in 1953 have remained unchanged.

Four key features define DNA structure

· Four features summarize the molecular architecture of DNA. (See Figure 11.6. The Instructor’s Resource CD-ROM includes a space-filling model of DNA.)

· The DNA molecule is a double-stranded helix.

· The diameter of the DNA molecule is uniform.

· The twist in DNA is right-handed (the twist is in the same direction as the threads on most screws).

· The two strands run in different directions (they are antiparallel).

· The sugar–phosphate backbones of each strand coil around the outside of the helix.

· The nitrogenous bases point toward the center of the helix.

· Hydrogen bonds between complementary bases hold the two strands together.

· A always pairs with T (two hydrogen bonds).

· G always pairs with C (three hydrogen bonds).

· Each base pair has one purine and one pyrimidine, so the diameter of the double helix remains constant.

· The direction of a polynucleotide is defined by the linkages between adjacent nucleotides. (See Figure 11.7.)

· The phosphate groups link the 3¢ carbon of one deoxyribose molecule to the 5¢ carbon of the next.

· Thus a single strand of DNA has a 5¢ phosphate group at one end (the 5¢ end) and a free 3¢ hydroxyl group at the other end (the 3¢ end).

· In a double helix, the 5¢ end of one polypeptide is hydrogen-bonded to the 3¢ end of the other, and vice versa.

The double helical structure of DNA is essential to its function

· The genetic material must perform four important functions:

· It must be able to store all of an organism’s genetic information.

· It must be susceptible to mutation.

· It must be precisely replicated in the cell division cycle.

· It must be expressible as the phenotype.

· The simple, double-helical structure of DNA, with the two strands linked by complementary base pairs, lends itself well to the first three of these functions.

· DNA is also well suited to expression as a phenotype, though this function is not inherent in the structure of the molecule.

DNA Replication

Three modes of DNA replication appeared possible

· Three years after Watson and Crick published their structure of DNA, the American biochemist Arthur Kornberg demonstrated that the DNA molecule contains the information needed for its own replication.

· Kornberg showed that DNA can replicate in a test tube with only a specific enzyme (DNA polymerase) and a mixture of four precursors: dATP, dCTP, dGTP, and dTTP.

· These precursors are deoxyribonucleoside triphosphates (dNTP’s).

· There is one precursor each for adenine, cytosine, guanine, and thymine.

· Theoretically, DNA could serve as its own template in one of three different ways:

· Semiconservative replication would use each parent strand as a template for a new strand. Each new DNA double helix would then have one parent strand and one new strand.

· Conservative replication would build an entirely new double helix based on the template of the old double helix. The new strand would contain none of the original DNA.

· Dispersive replication would use fragments of the original DNA molecule as templates for assembling two molecules. All the resulting strands would be mixtures of old and new material.

· Watson and Crick’s model suggested but did not prove that replication is semiconservative.

Meselson and Stahl demonstrated that DNA replication is semiconservative

· Matthew Meselson and Franklin Stahl demonstrated in 1957 that DNA replication is semiconservative by using a technique they devised called density labeling.

· Centrifuging in a cesium chloride (CsCl) solution can separate DNA labeled with “heavy” nitrogen (¹⁵N) from unlabeled DNA. (See Figure 11.9.)

· Meselson and Stahl grew a culture of E. coli for 17 generations in a medium with ¹⁵N instead of ¹⁴N.

· As a result, all the DNA in the bacteria was “heavy.”

· They then transferred bacteria grown on the heavy medium to a normal medium and allowed the bacteria to continue growing. (See Figure 11.10.)

· Under the conditions they used, E. coli replicates its DNA every 20 minutes.

· They sampled the DNA at each generation time, starting with the parental, all-heavy generation.

· Centrifuging the DNA after the first cell division (20 minutes) yielded a single band of DNA intermediate in density between the heavy and light forms.

· This ruled out the conservative replication hypothesis, which would have yielded two bands, one heavy and one light.

· Centrifuging the DNA after the second cell division (40 minutes) yielded an intermediate band and a light band, the result predicted by the semiconservative replication hypothesis.

· Dispersive replication would again have yielded a single band with a density less than heavy DNA but greater than light DNA.

· Other scientists demonstrated that DNA in eukaryotes also replicates semiconservatively.

The Mechanism of DNA Replication

· There are four requirements for semiconservative replication of DNA:

· DNA must act as a template for complementary base pairing.

· dATP, dGTP, dCTP, and dTTP must be present.

· The enzyme DNA polymerase must be present to bring the substrates to the template and catalyze the reactions.

· The reactions involved in replication are endergonic, so a source of chemical energy must be present.

· DNA replication takes place in two steps:

· The hydrogen bonds between the two strands are broken (the DNA is denatured), making each strand available for base pairing.

· The new nucleotides are covalently bonded to each growing strand.

· In virtually all DNA replication, nucleotides are added to the 3¢ end of the growing polynucleotide. (See Figure 11.11.)

· The three phosphate groups of the deoxyribonucleoside triphosphate are attached to the 5¢ position of the sugar.

· Energy for synthesis of nucleotides to the growing chain comes from breaking the bonds between these three phosphates.

· The free hydroxyl group at the 3¢ end of the growing chain reacts with one of the phosphate groups, breaking the bond between the phosphate group attached to the sugar and the two terminal phosphate groups.

· The breaking of this bond releases some energy for synthesis.

· The one phosphate group still attached to the 5¢ carbon of the new nucleotide bonds to the 3¢ end of the growing chain, becoming part of the sugar–phosphate backbone.

· Additional energy is released when the two freed phosphate groups (which constitute a pyrophosphate ion) break apart.

DNA is threaded through a replication complex

· A huge protein complex catalyzes the reactions of DNA replication.

· This replication complex recognizes an origin of replication on a chromosome.

· DNA replicates in both directions from the origin, forming two replication forks.

· In DNA replication, both strands of DNA act as templates.

· Until recently, it was believed that the replication complex moved along the strand of DNA. (See Figure 11.12.)

· Recent evidence suggests that the replication complex is stationary, and DNA threads through it.

· Replication complexes consist of several proteins with different roles.

· DNA helicase denatures the double helix.

· Single-strand binding proteins keep the two strands separate.

· RNA primase makes a primer strand that serves as a starting point for replication.

· DNA polymerase adds complementary nucleotides to the growing strand, proofreads the DNA, and repairs it.

· DNA ligase seals up breaks in the sugar–phosphate backbone.

Small, circular DNA’s replicate from a single origin, while large, linear DNA’s have many origins

· The enzyme DNA helicase uses energy from ATP to unwind the two DNA strands and make them available for complementary base pairing.

· Special proteins bind to the unwound strands to keep them apart.

· Small chromosomes, such as those found in bacteria, have a single origin of replication. (See Figure 11.13.)

· Replication in bacteria produces two interlocking circular DNA’s that are separated by an enzyme called DNA topoisomerase.

· Large chromosomes can have hundreds of origins of replication.

· Replication occurs at many different sites simultaneously.

DNA polymerases need a primer

· DNA polymerase is shaped like a hand with “finger” regions that rotate inward. (See Figure 11.14.)

· The finger regions have precise shapes that recognize the shapes of different bases.

· DNA polymerases cannot build a strand without having an existing strand of DNA or RNA, called a primer, to start from.

· In DNA replication, the primer strand is a short strand of RNA complementary to the DNA template strand. (See Figure 11.15.)

· An enzyme called a primase makes the primer strand.

· The primase is part of a protein complex called a primosome.

· The RNA primer is later degraded and replaced with DNA, so the final DNA molecule has no RNA.

DNA polymerase III extends the new DNA strands

· Bacterial cells contain more than one DNA polymerase.

· In prokaryotes, DNA polymerase III is responsible for elongation of new DNA strands.

· Recall that new bases are always added to the 3¢ end of a growing DNA strand.

· The strands in the template DNA are antiparallel, however.

· As a result, as the strands pass through the replication complex, one strand (the leading strand) will be in the correct orientation for addition of new nucleotides, but the other strand (the lagging strand) will be in the reverse orientation. (See Figure 11.17.)

The lagging strand is synthesized from Okazaki fragments

· Because of its backward orientation, the lagging strand must grow in relatively small, discontinuous pieces, called Okazaki fragments after their discoverer, the Japanese biochemist Reiji Okazaki.

· Each Okazaki fragment requires an RNA primer strand, which is formed by RNA primase some distance away from the previous Okazaki fragment. (See Figure 11.18.)

· DNA polymerase III synthesizes complementary DNA starting from the 3¢ end of the new primer and working toward the previous Okazaki fragment.

· When DNA polymerase III reaches the previous Okazaki fragment, it is released.

· DNA polymerase I then replaces the RNA primer of the previous Okazaki fragment with DNA.

· Finally, DNA ligase catalyzes formation of the phosphodiester linkage that joins the two Okazaki fragments.

· Okazaki fragments are 100 to 200 nucleotides long in eukaryotes, and 1,000 to 2,000 nucleotides long in prokaryotes.

· In E. coli, the replication complex makes new DNA at a rate in excess of 1,000 base pairs per second.

· Errors in replication are fewer than one base in a million.

DNA Proofreading and Repair

· Although errors in DNA replication (also known as mutations) are essential for evolution, the vast majority of DNA errors are neutral at best and fatal at worst.

· If DNA replication in humans were only as accurate as one base in a million, about 1,000 genes in every cell would be affected each time the cell divided.

· To minimize the number of errors, our cells normally have at least three DNA repair mechanisms at their disposal. (See Figure 11.19.)

· A proofreading mechanism corrects errors during the replication process.

· A mismatch repair mechanism scans and repairs errors in DNA shortly after replication.

· An excision repair mechanism operates over the life of the cell to repair errors that result from chemical or radiation damage.

Proofreading and repair mechanisms ensure that DNA replication is accurate

· As they add new bases to a growing strand, DNA polymerases make a proofreading check to make sure they have added the correct base.

· When a DNA polymerase recognizes an error, it removes the wrong nucleotide and tries again.

· Other proteins of the replication process also help out with this function.

· The error rate of DNA polymerase on each attempt is only about 1 in 10,000, so the second attempt at matching the template is very likely to be successful.

· This proofreading function reduces the overall error rate to about one base in a billion (one in 10⁹).

· The mismatch repair mechanism scans new DNA (following DNA replication and during genetic recombination) for mismatched base pairs.

· The mismatch repair mechanism operates before the new DNA strand is chemically modified (methylated).

· In eukaryotes, methyl groups are added some time after replication to some cytosines.

· In prokaryotes the methyl groups are added to guanine.

· This mechanism can distinguish between the methylated template strand and the unmethylated new strand.

· Thus, this mechanism can determine which base is correct (the base on the template strand) and which base needs to be replaced.

· One form of colon cancer arises in part from a failure of mismatch repair.

· Excision repair proteins operate over the life of a cell.

· Some cells live for many years, during which time their DNA is subject to damage by chemicals, radiation, and random spontaneous chemical reactions.

· Excision repair enzymes “inspect” the cell’s DNA for mispaired bases, chemically modified bases, and points where one strand has more bases than the other.

· These enzymes cut the damaged strand and remove the modified base and a few bases on either side of it.

· DNA polymerase and DNA ligase fill in and seal up the resulting gap.

· Various diseases can result from defects in the excision repair mechanism.

· The skin disease xeroderma pigmentosum results when the excision repair mechanism that repairs damage caused by ultraviolet radiation fails to work properly. People with this disease develop skin cancer very easily following exposure to sunlight.

DNA repair requires energy

· The energy for DNA synthesis comes primarily from the breaking of one of the high-energy bonds in the deoxyribonucleoside triphosphates (dNTP’s).

· The overall process of adding a dNTP to a growing strand, however, is slightly endergonic.

· Additional energy comes from cleaving of the pyrophosphate ion released from the dNTP.

· Hydrogen bonds between base pairs, as well as other weak interactions within the growing molecule, help drive the polymerization reaction.

· Thus, DNA synthesis itself does not require much energy beyond that stored in the substrate molecules.

· DNA repair, however, can require a lot of energy.

· The energy expended on DNA repair is an indication of how important the repair processes are.

Practical Applications of DNA Replication

· Two important laboratory techniques have been devised from the principles that underlie DNA replication: DNA sequencing and the polymerase chain reaction.

The nucleotide sequence of DNA can be determined

· The technique for sequencing DNA hinges on the difference between the normal substrates of DNA synthesis (deoxyribonucleoside triphosphates, or dNTP’s) and slightly modified substrate molecules (ddNTP’s). (See Figure 11.20a.)

· dNTP’s contain the sugar 2-deoxyribose.

· ddNTP’s contain the sugar 2,3-dideoxyribose.

· Like dNTP’s, ddNTP’s are picked up by DNA polymerase and added to a growing DNA chain.

· ddNTP’s lack a hydroxyl group at the 3¢ position, however, so no new nucleotide can be added after a ddNTP, and synthesis ends.

· Sequencing begins by denaturing a fragment of DNA, usually no more than 500 base pairs long. (See Figure 11.20b.)

· The single-stranded DNA is mixed with the following:

· DNA polymerase, for synthesis of complementary DNA strands

· Short primer strands, to help initiate synthesis

· The four normal dNTP substrates (dATP, dGTP, dCTP, and dTTP)

· Small amounts of the four ddNTP’s, each with a fluorescent tag to distinguish the different bases

· In solution, DNA polymerase synthesizes strands of DNA using mostly the normal dNTP substrates.

· When DNA polymerase encounters a ddNTP, chain growth stops.

· The result is a solution with template DNA strands and shorter complementary strands, each one ending with a fluorescently tagged ddNTP.

· The new strands are denatured from the templates and separated by electrophoresis (see Figure 17.2).

· This technique orders the strands by length and can distinguish strands that differ in length by only one base.

· The shortest fragments (which travel farthest in the electrophoresis) should be just one base longer than the primer strand.

· The color of the fluorescent tag at the end of this sequence indicates the type of ddNTP that was added.

· If this was ddATP, for example, then the first base on the template strand (after the primer sequence) is T.

· The remainder of the bases on the template strand can be determined in a similar manner.

· This process has been automated with computers, which can also analyze the sequence.

· These analyses have formed the basis of the new science of genomics.

The polymerase chain reaction makes multiple copies of DNA

· The polymerase chain reaction (PCR) technique is a simple method for making multiple copies of a DNA sequence. (See Figure 11.21.)

· PCR cycles through three steps:

· Heat double-stranded DNA to denature it into single strands.

· Add short primer strands specific for sequences at the 3¢ ends of the complementary strands.

· Add DNA polymerase to synthesize new DNA.

· A single cycle takes only a few minutes and doubles the amount of DNA.

· With enough primer, DNA polymerase, and substrate dNTP’s, repeating the cycle many times leads to a geometric increase in the number of copies of DNA.

· The primer strands, usually 15 to 20 bases long, must be made in the laboratory.

· This requires sequencing the first 15 to 20 bases at the 3¢ end of each complementary strand.

· It is unlikely that strands of this length will bind to more than one location on the target DNA strand.

· PCR did not become practical until the discovery of a DNA polymerase that could survive the heat required to denature the DNA.

· Such a DNA polymerase was found in bacteria that live in hot springs at Yellowstone National Park. (Chapter 26 of the Instructor’s Resource CD-ROM includes a photograph of a geyser at Yellowstone National Park.)

· The biochemist Kerry Mullis earned a Nobel prize for applying the DNA polymerase from thermophilic bacteria to the PCR technique.

· PCR has had an enormous impact on genetic research.

The Instructor’s Resource CD-ROM includes a space-filling model of DNA and an X-ray diffraction pattern of DNA.