DNA and Its Role in Heredity
DNA and Its Role in Heredity
DNA: The Genetic Material
The Structure of DNA
Determining the DNA Replication Mechanism
The Molecular Mechanisms of DNA Replication
DNA Proofreading and Repair
Practical Applications of DNA Replication
DNA: The Genetic Material
By the early twentieth century, geneticists had associated the presence of
genes with chromosomes.
Circumstantial evidence pointed to DNA as the ________ material.
DNA was found in the nucleus and chromosomes, which were already known to
carry genes.
A dye that binds to DNA showed that the amount of DNA in ________ cells was
________ that in eggs or sperm, as would be expected from Mendel's discoveries.
DNA: The Genetic Material
In the 1920s, the English physician Frederick Griffith did experiments with
two strains of Streptococcus pneumoniae.
He discovered that a chemical ________ principle from one strain could cause a
heritable change in the other strain.
DNA: The Genetic Material
Oswald T. Avery and colleagues spent several years identifying the
transforming principle.
They treated the extract from the S-strain bacteria in various ways to destroy
different types of substances but retain others.
When DNA was destroyed, the transforming activity was lost, but when DNA was
left intact, the ________ activity survived.
DNA: 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.
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.
DNA: The Genetic Material
Some viruses were labeled with radioactive ________ which is present in
proteins but not in DNA.
Other viruses were labeled with radioactive ________ which is present in DNA
but absent from most proteins.
The labeled sulfur (and thus the viral protein) separated from the bacteria,
but the labeled phosphorus (and thus the viral DNA) remained with the bacteria.
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?
The structure of DNA was determined after many types of evidence were
combined.
The Structure of DNA
The positions of atoms in a crystalline substance can be inferred from the
pattern of diffraction of X-rays passed through it.
In the early 1950s, many skilled X-ray crystallographers tried but failed to
glean information from X-ray diffraction patterns of DNA.
The English chemists Rosalind Franklin and Maurice Wilkins were able to
provide key information about the structure of DNA based on X-ray
crystallography.
The Structure of DNA
By the 1950s it was known that DNA was a polymer of nucleotides.
The four nucleotides that make up DNA differ only in their nitrogenous bases.
There are two________ (adenine and guanine) and two pyrimidines (cytosine and
thymine).
In 1950, Erwin ________ 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.
The Structure of DNA
English physicist Francis Crick and American geneticist James D. Watson
established 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 ________ to each other.
The Structure of DNA
Four features summarize the molecular architecture 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 two strands run in different directions (they are antiparallel).
The Structure of DNA
The sugarphosphate backbones of each strand coil around the outside of the
helix.
The nitrogenous bases point toward the center of the helix.
Hydrogen bonds between ________ bases hold the two strands together.
A always pairs with T (two hydrogen bonds).
G always pairs with C (three hydrogen bonds).
The Structure of DNA
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 Structure of DNA
The genetic material performs four important functions:
§ It stores all of an organism's genetic information.
§ It is susceptible to mutation.
§ It must be precisely replicated in the cell division cycle.
§ It is expressed as the phenotype.
Determining the DNA Replication Mechanism
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 (deoxyribonucleoside
triphosphates): dATP, dCTP, dGTP, and dTTP.
Determining the DNA Replication Mechanism
Theoretically, DNA could serve as its own template in one of ________
different ways:
§ Semiconservative replication would use each parent strand as a template for a
new strand.
§ Conservative replication would build an entirely new double helix based on the
template of the old double helix.
§ Dispersive replication would use fragments of the original DNA molecule as
templates for assembling two molecules.
Determining the DNA Replication Mechanism
Matthew ________ and Franklin ________ demonstrated in 1957 that DNA
replication is semiconservative by using a technique called density labeling.
They used DNA labeled with "heavy" nitrogen (15N).
The Molecular Mechanisms of DNA Replication
DNA replication takes place in two steps:
§ The hydrogen bonds between the two strands are broken, making each strand
available for base pairing.
§ The new nucleotides are covalently bonded to each growing strand.
The Molecular Mechanisms of DNA Replication
In DNA replication, nucleotides are added to the 3’ end of the growing strand.
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 Molecular Mechanisms of DNA Replication
A huge protein complex catalyzes 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.
Recent evidence suggests that the ________ ________ is stationary, and DNA
threads through it.
The Molecular Mechanisms of DNA Replication
The enzyme DNA ________ uses energy from ATP to unwind the two DNA strands.
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.
Replication in bacteria produces two interlocking circular DNAs that are
separated by the enzyme DNA topoisomerase.
The Molecular Mechanisms of DNA Replication
Large chromosomes can have ________ of origins of replication.
Replication occurs at many different sites simultaneously.
The Molecular Mechanisms of DNA Replication
DNA polymerases cannot build a strand without having an existing strand,
called a primer, to start from.
In DNA replication, the ________ strand is a short strand of RNA complementary
to the DNA template strand.
An enzyme called a ________ makes the primer strand.
The primase is part of a protein complex called a primosome.
The Molecular Mechanisms of DNA Replication
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.
The Molecular Mechanisms of DNA Replication
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.
The other strand (the lagging strand) will be in the reverse orientation.
The Molecular Mechanisms of DNA Replication
Because of its backward orientation, the lagging strand must grow in
relatively small, discontinuous pieces, called ________ fragments.
Each Okazaki fragment requires an RNA primer strand, which is formed by RNA
primase.
DNA polymerase III synthesizes complementary DNA starting from the 3’ end of
the new primer and working toward the previous Okazaki fragment.
The Molecular Mechanisms of DNA Replication
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.
The Molecular Mechanisms of DNA Replication
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 ________
to bind.
New chromosomes formed after DNA replication have single-stranded DNA at each
end.
This single-stranded region is cut off, slightly ________ the chromosome after
each cell division.
The Molecular Mechanisms of DNA Replication
Many eukaryotic chromosomes have repetitive sequences called ________ at their
ends that shorten after each round of cell division.
After a given number of cell divisions, the telomeres have shortened to the
extent that they are no longer able to ________ the ends of the chromosomes, and
no cell division can occur.
This results in cell death and explains in part why cells do not last the
entire lifetime of the organism.
The Molecular Mechanisms of DNA Replication
Constantly dividing cells, such as bone marrow, germ line, and more than 90
percent of cancer cells, produce an enzyme called ________ that catalyzes the
addition of any lost telomeric sequences.
DNA Proofreading and Repair
Although errors in DNA replication (mutations) are essential for evolution,
the vast majority of DNA errors are ________ at best and ________ at worst.
To minimize the number of errors, our cells have three DNA repair mechanisms:
§ Proofreading
§ Mismatch repair
§ Excision repair
DNA Proofreading and Repair
As they add new bases to a growing strand, DNA polymerases make a proofreading
check.
When a DNA polymerase recognizes an error, it removes the wrong nucleotide and
tries again.
The error rate of DNA polymerase on each attempt is only about 1 in 10,000.
This proofreading function reduces the overall error rate to about one base in
a billion.
DNA Proofreading and Repair
The mismatch repair mechanism scans new DNA for mismatched base pairs.
The mismatch repair mechanism operates before the new DNA strand is
methylated.
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.
DNA Proofreading and Repair
Excision repair proteins operate over the life of a cell.
DNA is subject to damage by chemicals, radiation, and random spontaneous
chemical reactions.
Excision repair enzymes "inspect" the cell's DNA for damage, then cut the
damaged strand and remove it.
DNA polymerase and DNA ligase fill in and seal up the resulting gap.
Practical Applications of DNA Replication
The polymerase chain reaction (PCR) technique is a simple method for making
multiple copies of a DNA sequence.
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.
Practical Applications of DNA Replication
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 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.
Practical Applications of DNA Replication
PCR did not become practical until the discovery of a DNA ________ 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 this technique.
PCR has had an enormous impact on genetic research.
Practical Applications of DNA Replication
The technique of DNA sequencing hinges on the use of modified nucleosides
(ddNTPs).
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.
Practical Applications of DNA Replication
Sequencing begins by denaturing a fragment of DNA.
The single-stranded DNA is mixed with DNA polymerase, short primer strands,
the four normal dNTP substrates, and small amounts of the four ddNTPs, each with
a fluorescent tag.
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.
Practical Applications of DNA Replication
The new strands are ________ from the templates and separated by ________ a
technique that separates strands by length.
The shortest fragments should be 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.
Animation 11.1 The Meselson-Stahl Experiment
Animation 11.2 DNA Replication, Part 1: DNA Polymerization
Animation 11.2 DNA Replication, Part 1: Replication of a Chromosome
Animation 11.3 DNA Replication, Part 2: Coordination of Leading and Lagging
Strand Synthesis
Video 11-01