Download Introduction - Cedar Crest College

DNA: The Genetic Material
•
By the early twentieth century, geneticists had associated the presence of genes with chromosomes and had
begun researching which chemical component of chromosomes comprised this genetic material.
•
Circumstantial evidence pointed to DNA as the genetic material.
•
DNA was found in the nucleus and chromosomes, which were 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.
•
The DNA-binding dye also showed that the amount of DNA varied among species. Each species appeared to
have its own specific nuclear DNA content.
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, and 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 did kill the mice, however.
•
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 heatkilled 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 for two reasons.
•
Most scientists did not believe that DNA was chemically complex enough to be the genetic material.
•
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.)
•
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 (protein or DNA) entered the bacterium.
(See Figure 11.3.)
•
To trace the two components of the virus over its life cycle, Hershey and Chase labeled each with a specific
radioactive tracer.
•
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?
(See Video 11.1.)
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.)
•
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 3.24 and 3.25.)
•
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. (See Figure 11.6a.)
•
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 11.6b.)
•
The DNA molecule is a double-stranded helix.
•
The diameter of the DNA molecule is uniform.
•
The twist in DNA is right-handed (like 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.
Determining the DNA Replication Mechanism
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 (dNTPs).
•
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. (See Figure 11.8a.)
•
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. (See Figure 11.8b.)
•
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. (See Figure 11.8c.)
•
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. (See Figure 11.9. and Animated Tutorial 11.1.)
•
Centrifuging can separate DNA labeled with “heavy” nitrogen (15N) from unlabeled DNA.
•
Meselson and Stahl grew a culture of E. coli for many 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.
•
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 Molecular Mechanisms of DNA Replication
•
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 11.10.)
•
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
•
•
•
•
•
11.11.)
•
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
Recent evidence suggests that the replication complex is stationary, and DNA threads through it.
Small, circular DNAs replicate from a single origin
•
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 11.12a.)
•
Replication in bacteria produces two interlocking circular DNAs that are separated by the enzyme DNA
topoisomerase.
Large, linear DNAs have many origins
•
•
Large chromosomes can have hundreds of origins of replication.
Replication occurs at many different sites simultaneously. (See Figure 11.12b.)
DNA polymerases need a primer
•
DNA polymerase is shaped like a hand with “finger” regions that rotate inward. (See Figure 11.13.)
•
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 11.14.)
•
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.
Cells contain several different DNA polymerases
•
Most cells contain more than one DNA polymerase.
•
Only one of the polymerases is responsible for chromosomal DNA replication.
•
The others are involved in primer removal and DNA repair.
•
See Figure 11.15 to examine some of the other proteins that collaborate at the replication fork.
•
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 11.16.)
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 11.17 and Animated Tutorial 11.3.)
•
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.
Telomeres are not fully replicated
•
•
Recall that replication of the lagging strand occurs by the addition of Okazaki fragments to RNA primers.
Beyond the very end of a linear DNA molecule, there is no place for a primer to bind.
•
This means that new chromosomes formed after DNA replication have single-stranded DNA at each end.
(See Figure 11.18a.)
•
This single-stranded region is cut off, along with some of the intact double-stranded end, slightly shortening
the chromosome after each cell division.
•
Many eukaryotic chromosomes have repetitive sequences called telomeres at their ends that shorten after
each round of cell division. These repeats bind to special proteins that maintain the stability of the chromosome ends.
(See Figure 11.18c.)
•
After a given number of cell divisions (20 to 30 in some human cells), the telomeres have shortened to the
extent that they are no longer able to stabilize the ends of the chromosomes, and the chromosomes are unable to take
place in cell division.
•
This results in cell death and explains in part why cells do not last the entire lifetime of the organism.
•
Constantly dividing cells, such as bone marrow, germ line, and more than 90 percent of cancer cells, produce
an enzyme called telomerase that catalyzes the addition of any lost telomeric sequences. (See Figure 11.18b.)
•
(See Animated Tutorial 11.2.)
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 results in one error for each million bases replicated, 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 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 10 9).
Mismatch repair mechanisms correct base pairing errors
•
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 mechanisms repair chemical damage
•
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.
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 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 11.20.)
•
PCR cycles through three steps:
•
Double-stranded fragments of DNA are heated to denature them into single strands.
•
A short primer is added, along with the four dNTPs.
•
DNA polymerase catalyzes the production of new DNA strands.
•
A single cycle takes only a few minutes and doubles the amount of DNA.
•
With enough primer, DNA polymerase, and substrate dNTPs, repeating the cycle many times leads to a
geometric increase in the number of copies of DNA.
•
The primer strands, usually 15–20 bases long, must be made in the laboratory.
•
This requires sequencing the first 15–20 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.
•
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 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 dNTPs) and slightly modified substrate molecules (ddNTPs). (See Figure
11.21a.)
•
dNTPs contain the sugar 2-deoxyribose.
•
ddNTPs contain the sugar 2,3-dideoxyribose.
•
Like dNTPs, ddNTPs are picked up by DNA polymerase and added to a growing DNA chain.
•
ddNTPs 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 700 base pairs long. (See Figure
11.21b.)
•
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 ddNTPs, 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, a technique that orders
the strands by length and can distinguish strands that differ in length by only one base. (See Figure 16.2.)
•
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.