Chapter 14: The nature of the genetic material
14.1/ the nature of the genetic material
Griffith finds that bacterial cells can be transformed
Discovery of transformation in bacteria
Frederick Griffith, 1928 studied Streptococcus pneumoniae, a pathogenic bacterium causing
pneumonia.
There are 2 strains of Streptococcus:
- S strain is the normal virulent form
- R strain is the mutant non-virulent form
Griffith infected mice with these strains and discovered transformation = process of transferring
“information” specifying virulence from the dead S strain cells into the live R strain cells.
Discovery of DNA as the transforming material in bacteria
[Link] and co-workers [Link], & [Link], 1944
they repeated Griffith’s experiment using purified cell extracts and discovers:
- Removal of all proteins and lipids from the transforming material did not destroy its
ability to transform R strain cells
- DNA-digesting enzymes destroyed all transforming ability
- The transforming material is DNA
Discovery of DNA as the phage genetic material
[Link] & [Link], 1952
- investigated bacteriophages = phages = viruses that infect bacteria
- the bacteriophage was composed of only DNA and protein (lytic phage)
- they wanted to determine which of these molecules is the genetic material that is injected
into the bacteria.
if they could identify the molecule injected into the cell, they would identify the viruses’
genetic material. To do this, they needed a method to introduce unique labels into both DNA
and protein. Nucleotides contain phosphorus, but proteins do not, and some amino acids
contain sulfur, but DNA does not. Thus, the radioactive 32P isotope will label DNA specifically,
and the isotope 35S will label proteins specifically. The two isotopes are easily distinguished
based on the particles they emit when they decay.
two experiments were performed. In one, viruses were grown on. A medium containing 32P,
which was incorporated into DNA; in the other, viruses were grown on medium containing 35S,
which was incorporated into coat proteins. Each culture of labeled viruses was then used to
infect separate bacterial cultures.
After infection, the bacterial cell suspension was agitated in a blender to remove the infecting
viral particles from the surfaces of the bacteria. This step ensured that only the part of the virus
that had been injected into the bacterial cells- that is, the genetic material- would be detected.
Each bacterial suspension was then centrifuged to produce a pellet of cells for analysis. In the
32
P experiment, a large amount of radioactive phosphorus was found in the cell pellet, but in
the 35S experiment, very little radioactive sulfur was found in the pellet. Hershey and chase
deduced that the genetic information viruses injected into the bacteria was DNA, and not
protein.
14.2/ DNA structure
DNA was discovered in 1869 by Friedrich Miescher. He extracted a white substance from the
nuclei of white blood cells. He also isolated the same substance from salmon sperm. Analysis of
this material, which was rich in phosphorus, indicated that it appeared to differ from other
known cellular constituents. Miescher named this new biological substance “nuclein” because it
was associated with the nucleus. Later, because nuclein was slightly acidic, it came to be called
nucleic acid.
DNA’s components were known, but its three-dimensional structure was a mystery
The components of nucleic acids were known:
- A 5-carbon sugar
- A phosphate (PO4-) group
- A nitrogen-containing (nitrogenous base). This may be a two-ringed purine (adenine, A,
or guanine, G), or a single-ringed pyrimidine (thymine, T, or cytosine, C); in RNA the
pyrimidine uracil, U, replace thymine, T)
The phosphate group is attached to the 5’ carbon atom of the sugar, and the bases are
attached to the 1’ carbon. In addition, a free hydroxyl (-OH) group is attached to the 3’
carbon atom. This means each nucleotide has a 5’ phosphate end, and a 3’ hydroxyl end.
Nucleotide monomers are joined together by a dehydration reaction involving the 5’
phosphate of one nucleotide with the 3’ hydroxyl of another nucleotide. This linkage is
called a phosphodiester bond because the phosphate group is now linked to the two sugars
by means of a pair of ester bonds.
Discovery of the 3D structure of DNA
Combined work of scientists:
- Erwin Chargaff determined that
A. Amount of adenine = amount of thymine
B. Amount of cytosine = amount of guanine
C. A + G = T + C
this is known as Chargaff’s rule
X-ray diffraction patters of DNA
- Rosalind Franklin and Maurice Wilkins, 1953
- Franklin performed X-ray diffraction studies to identify the 3D structure
- He discovered that DNA is helical: diameter of 2 nm, makes a complete turn of helix
every 3.4 nm
Generation of a 3D model of DNA structure
- James Watson and Francis Crick, 1953
- Deduced the 3D structure of DNA using evidence from Chargaff, Franklin, and others
A. 2 sugar-phosphate backbones – phosphodiester backbone-
B. Nitrogenous bases toward the interior of the molecule
C. Bases from hydrogen bonds with complementary bases on the opposites sugar-
phosphate backbone
D. The two strands of nucleotides are antiparallel to each other: one is oriented 5’ to 3’,
the other 3’ to 5’.
E. The two strands wrap around each other to create the helical shape of the molecule =
double helix
14.3/ Basic characteristics of DNA replication
Meselson and Stahl demonstrated the semiconservative mechanism
In replication, the sequence of parental strands must be duplicated in daughter strands. That is,
one parental helix with two strands must yield two daughter helices with four strands. The two
daughter molecules are then separated during the course of cell division:
Three models of DNA replication are possible:
1. In a conservative model, both strands of the parental duplex would remain intact
(conserved), and new DNA copies would consist of all-new molecules. Both daughter
strands would contain all-new molecules.
2. In a semiconservative model, one strand of the parental duplex would remain intact in
each daughter duplex (semiconserved); a new complementary strand would be
synthesized with each parental strand. Each daughter duplex would consist of one
parental strand and one newly synthesized strand.
3. In a dispersive model, copies of DNA would consist of mixtures of parental and newly
synthesized strands; that is, the new DNA would be dispersed throughout each strand of
both daughter molecules after replication.
The Meselson-Stahl experiment:
Bacterial cells were grown in a medium containing 15N, which became incorporated into the
bases of the bacterial DNA. After several generations, the DNA of these bacteria was denser
than that of bacteria grown in a medium containing the normally available 14N. Meselson and
Stahl then transferred the bacteria from the 15N medium to 14N medium and collected the DNA
at various time intervals.
Interpretation:
1. The conservative model was not consistent with the data because after one round of
replication, two densities should have been observed: DNA strands would either be all-
heavy (parental) or all-light (daughter). This model is rejected.
2. The semiconservative model is consistent with all observations: after one round of
replication, a single density would be predicted because all DNA molecules would have
light strand and a heavy strand. After two round of replication, half of the molecules
would have two light strands, and half would have a light strand and a heavy strand- and
so two densities would be observed. Therefore, the results support the
semiconservative model.
3. The dispersive model was consistent with the data from the first round of replication,
because in this model, every DNA helix would consist of strands that are mixture of ½
light (new) and ½ heavy molecules. Instead, two densities were observed. Therefore,
this model is also rejected.
The basic mechanism of SNA replication is semiconservative. At the simplest level, then,
DNA is replicated by opening up a DNA helix and making copies of both strands to produce
two daughter helices, each consisting of one old strand and one new strand.
DNA replication: material and steps
DNA replication requires:
- Templates
- Enzymes
- Nucleotides: dATP , dGTP, dTTP and dCTP
DNA replication includes three steps:
- Initiation – replication begins at an origin of replication
- Elongation – new strands of DNA are synthesized by DNA polymerase
- Termination – replication is terminated differently in prokaryotes and eukaryotes
All DNA polymerases that have been examined have several common features. They all add
new bases to the 3’ end of existing strands. That is, they synthesize in a 5’ to 3’ direction by
extending a strand base-paired to the template. All DNA polymerases also require a primer to
begin synthesis; they cannot begin without a strand of RNA or DNA base-paired to the
template. RNA polymerases do not have this requirement, so they usually synthesize the
primers.
14.4/ Prokaryotic Replication
We use [Link] as a model system.
- It has a small genome
- It has a large collection of mutants that affect replication
- It is a circular molecule of DNA = 1 chromosome
Prokaryotic replication starts at a single origin
In [Link], replication begins at a specific origin called oriC when a protein called DnaA recognizes
and binds to oriC. This causes an AT-rich sequence to be melted, making single strands available
to load the enzymatic machinery for replication. An AT-rich region is easir to melt, as A-T base-
pairs have only two hydrogen bonds compared to the three in G-C base-pairs.
After initiation, replication proceeds bidirectionally from this unique origin to the unique
terminus. We call the DNA controlled by an origin a replicon. In this case, the chromosome plus
the origin forms a single replicon.
[Link] has at least three different DNA polymerases
DNA polymerase refers to a class of enzymes that use a DNA template to assemble a new
complementary strand. The first DNA polymerase isolated in [Link] was DNA polymerase I (Pol
I). At first, investigators assumed this polymerase was all that was required for DNA replication.
Later, a mutant was isolated with Pol I activity, but that could still replicate its chromosome.
Two additional polumerases were isolated from this strain of [Link]: DNA polymerase II (Pol II)
and DNA polymerase III (Pol III). As we all know DNA polymerases, all three of these enzymes
synthesize polynucleotide strands only in the 5’ to 3’ direction and requires a primer.
In addition to adding nucleotides to a growing DNA strand, some polymerases can also remove
nucleotides, or act as a nuclease. Enzymes that act as nucleases are classified as either
endonuclease (which cut DNA internally) or exonucleases (which remove nucleotides from the
end of DNA). DNA Pol I, Pol II, and Pol III have 3’ to 5’ exonuclease activity, which serves as a
proofreading function because it allows the enzyme to “back up” and remove a mis paired
base. DNA Pol I also have a 5’ to 3’ exonuclease activity, which can be used to remove RNA
primers.
The three different polymerases have different roles in the replication process. DNA Pol III is the
main replication enzyme; it is responsible for the bulk of DNA synthesis. DNA Pol I act on the
lagging strand to remove primers and replace them with DNA. The Pol II enzyme does not
appear to play a role in replication but it involved in DNA repair process.
Unwinding DNA requires energy and causes torsional strain
Although most DNA polymerases can unwind DNA during synthesis, replication is more efficient
if the helix is unwound ahead of the polymerase. An enzyme with DNA unwinding activity is
called a helicase. This process requires energy in the form of ATP, and can progressively unwind
DNA, forming single strands. These single strands are not stable because the hydrophobic bases
are exposed to water. Cells solve this problem with another protein, single-stranding-binding
protein (SSB), that will coat exposed single strands.
The unwinding of the two strands introduces torsional strain in the DNA molecule. Imagine two
rubber bands twisted together. If you know unwind the rubber bands, what happens? The
rubber bands, already twisted about each other, will further coil in space. When this happens
with a DNA molecule it is called supercoiling.
Enzymes that alter the topological state of TNA are called topoisomerases. Topoisomerase
enzymes act to relieve the torsional strain caused by unwinding and to prevent this supercoiling
from happening. DNA gyrase is the topoisomerase involved in DNA replication.
DNA primase sensitizes short stretches of RNA ( 10 to 20 base-pairs) that serve as primers for
Pol III.
Replication is semidiscontinuous
Earlier, DNA was described as being antiparallel – meaning that one strand runs in the 3’ to 5’
direction, and its complementary strand runs in the 5’ to 3’ direction. The antiparallel nature of
DNA combined with the nature of the polymerase enzymes puts constraints on the replication
process. Because polymerases can synthesize DNA in only one direction, and the two DNA
strands run in opposite directions, polymerases on the two strands must be synthesizing DNA in
opposite directions.
The requirement of DNA polymerases for a primer means that on one strand primers need to
be added as the helix opened up. This means that one strand can be synthesized in a
continuous fashion from an initial primer, but the other strand must be synthesized in a
discontinuous fashion with multiple priming events and short sections of DNA being assembled.
The strand that is continuous is called the leading strand, and the strand that is discontinuous is
the lagging strand. DNA fragments synthesized on the lagging strand are named Okazaki
fragments. They introduce a need for even more enzymatic activity on the lagging strand.
- Pol III can only add nucleotides to the 3’ end of the newly synthesized strand
- Two strands serve as templates to synthesize DNA but in opposite directions
- Replication fork = two single strands of DNA have a fork appearance after partial
denaturation
- DNA ligase seals the ‘nicks’ and joins the Okazaki fragments into complete strands.
Leading strand synthesis
Synthesis on the leading strand is relatively simple. A single priming event is required, and Then
strand can be extended indefinitely by the action of DNA Pol III. If the enzyme remains attached
to the template, it can synthesize around the entire circular E. coli chromosome.
The ability of a polymerase to remain attached to the template is called processivity. The Pol III
enzyme is a large multisubunit enzyme that has high processivity due to the action of one
subunit of the enzyme, called the beta subunit.
The beta subunit is made of two identical protein chains that come together to form a circle.
This circle can be loaded onto the template like a clamp to hold the Pol III enzyme to the DNA.
This structure is therefore referred to as the “sliding clamp”, and a similar structure is found in
eukaryotic polymerases as well. For the clamp to function, it must be opened and then closed
around the DNA. A mutisubunit protein called the clamp loader accomplished this task. This
function is also found in eukaryotes.
Lagging strand synthesis
Because synthesis on the lagging strand is discontinuous, more steps are required to replicate
this strand. Primase is needed to synthesize primers for each Okazaki fragment, and then all
these RNA primers must be removed and replaced with DNA. Finally, the fragments need to be
stitched together.
The Okazaki fragments themselves are synthesized by DNA Pol III, like the leading strand, but
the primers are removed and replaced with DNA by DNA Pol I. with its 5’ to 3’ exonuclease
activity, Pol I can remove primers and then replace them with its unusual polymerase activity.
The Pol I extend the Okazaki fragment “behind” it, while removing the RNA primer “in front of”
it.
This leaves only the last phosphodiester bond to be formed where synthesis by Pol I ends. This
is done by DNA ligase, which seals this “nick”, acting to join the Okazaki fragments into
complete strands.
Termination
Termination occurs at a specific site located roughly opposite oriC on the circular chromosome.
The last stages of replication produce two daughter molecules that are intertwined like two
rings in a chain. These intertwined molecules are unlinked by the same enzyme that relieved
torsional strain at the replication fork: DNA gyrase.
The replisome contains all the necessary enzymes for replication
Cells have many macromolecular machines that perform specific functions, such as the
ribosome that synthesizes proteins. The macromolecular machine responsible for DNA
replication is the replisome. The replisome has two main subcomponents: the primosome
consist of primase and helicase, along with a number of accessory proteins.
Despite our calling one strand the lagging strand, the two Pol III enzymes in the replisome are
active on both strands simultaneously. How can the two strands be synthesized in the same
direction when strands are antiparallel? The best model involves a loop formed in the lagging
strand, allowing the two polymerases to move in the same direction.
The two Pol III complexes include two synthetic core subunits, each with its own beta sliding
clamp subunit. The entire replisome complex is held together by a number of proteins,
including the clamp loader. As Pol III finished an Okazaki fragment, the clamp loader loads a
beta subunit onto the next fragment, and transfers the Pol III to this new beta subunit.
14.5/ Eukaryotic Replication
Eukaryotic replication is complicated by two main factors: the larger amount of DNA organized
into multiple chromosomes, and the linear structure of the chromosomes. This process requires
new enzymatic activities only for dealing with the ends of chromosomes, otherwise the basic
enzymology is the same.
Eukaryotic replication uses multiple origins
The sheer amount of DNA and how it is packaged constitute a problem for eukaryotes.
Eukaryotes usually have multiple chromosomes that are each larger than the E. coli
chromosome. If each chromosome had a single origin, the time necessary for replication would
be prohibitive. Eukaryotic cells reduce the time needed for replication by using multiple origins
for each chromosome, resulting in multiple replicons. The origins are not as sequence-specific
as oriC, and their recognition seems to depend on chromatin structure as well as on sequence.
The number of origins used can also be adjusted during the course of development, so. That
early on, when cell divisions need to be rapid, more origins are activated. Each origin must be
used only per cell cycle.
The eukaryotic replication fork is more complex
Prior to S phase, helicase complexes are loaded onto possible replication origins, but not
activated. Then, during S phase a subset of these are activated, and the rest of the replisome
assembled. The action of the replisome differs from that in E. coli in a few ways we will
highlight.
Priming is accomplished by a complex of DNA polymerase alpha and primase. This complex
synthesizes primers consisting of RNA and a short stretch of DNA. There are also different
polymerases for the leading and lagging strands. DNA polymerase epsilon (Pol E) is responsible
for leading strand synthesis and DNA polymerase delta does the lagging strand. The sliding
clamp subunit that allows the enzyme complex to stay attached to the template is called PCNA
(for proliferating cell nuclear antigen). The name comes from its identification as an antibody-
inducing protein in proliferating (dividing) cells. The PCNA sliding clamp forms a trimer, but has
a similar structure and function to the beta subunit sliding clamp.
Synthesis on the lagging strand differs most from related events in prokaryotic DNA replication.
The Okazaki fragments are significantly shorter. As each fragment reaches the previous
fragment, synthesis continues to displace the strand containing the previous primer. The
displace primer is removed by a nuclease, and the remaining fragments joined by ligase.
Archaeal and eukaryotic replication proteins are evolutionarily related
The replisome, which can replicate both strands of DNA simultaneously, requires a variety of
functions, including DNA unwinding (helicase), synthesis (primase and polymerase), and
processivity factors (sliding clamps and their loaders). There is clear functional conservation of
these activities across all domains of life, but major components appear to have evolved
independently in bacteria, and in archaea/eukarya.
Members of the DNA polymerase family of enzymes have similar structure and homologous
synthetic domains. They are differentiated based on nonhomologous regions into seven
subfamilies. The subfamily used by eukaryotes and archaea is the same, but different from the
one used by bacteria. Replicative helicases all have a similar structure, forming a ring around a
single strand of DNA, but the bacterial and achaeal/eukaryal enzymes are not homologous, and
even move in the opposite direction along DNA. Lastly, the primase component of the
replisome is also not homologous between bacteria and archaea/eukarya. The one function
that is clearly homologous in all domains of life is the processivity factors: sliding clamps and
clamp loaders.
Linear chromosomes have specialized ends
The specialized structures found on the ends of eukaryotic chromosomes are called telomeres.
These structures protect the ends of chromosomes from nucleases and maintain the integrity
of linear chromosomes. These telomeres are composed of specific DNA sequences, but they are
not made by the replication complex.
Replicating ends
The very structure of a linear chromosome causes a cell problem in replicating the ends. The
directionality of polymerases, combined with their requirement for a primer, creates this
problem. Consider a simple linear molecule. Replication of one end of each template strand is
simple, namely the 5’ end of the leading-strand template. When the polymerase reaches this
end, synthesizing in the 5’ to 3’ direction, it eventually runs out of template and is finished.
But on the other strand’s end, the 3’ end of the lagging strand, removal of the last primer on
this end leaves a gap. This gap cannot be primed, meaning that the polymerase complex cannot
finish this end properly. The result would be gradual shortening of chromosomes with each
round of cell division.
The action of telomerase
When the sequence of telomeres was determined, they were found to be composed of short
repeated sequences of DNA. This repeating nature is easily explained by their synthesis. They
are made by an enzyme called telomerase, which uses an internal RNA as a template and not
the DNA itself. The use of the internal RNA template allows short stretches of DNA to be
synthesized, composed of repeated nucleotide sequences complementary to the RNA of the
enzyme. The other strand of these repeated units is synthesized by the usual action of the
replication machinery copying the strand made by telomerase.
Telomerase, aging, and cancer
A gradual shortening of the ends of chromosomes occurs in the absence of telomerase activity.
During embryonic and childhood development in humans, telomerase activity is high, but is low
in most somatic cells of the adult. The exceptions are cells that must divide as part of their
function, such as lymphocytes. The activity of telomerase in somatic cells is kept low by
preventing the expression of the gene encoding this enzyme.
To examine the role of telomerase, mice were produced that completely lacked telomerase
activity. These mice appeared to be normal for up to six generations, but they showed steadily
decreasing telomere length that eventually lead to nonviable offspring. This implies a
relationship between cell senescence (aging) and telomere length.
Normal cells undergo only a specified number of divisions when grown in culture. This limit at
least partially based on telomere length. Support for this relationship between senescence and
telomere length comes from experiments in which telomerase was introduced into fibroblasts
in culture. These cells had their life span increased relative to controls that had no added
telomerase. Interestingly, these cells did not show the hallmarks of malignant cells, indicating
that activation of telomerase alone does not make cells malignant.
A relationship has been found, however, between telomerase and cancer. Cancer cells do
continue to divide indefinitely, and this would not be possible if their chromosomes were being
continually shortened. Cancer cells generally show activation of telomerase which allows them
to maintain telomere length; but this is clearly only one aspect of conditions that allow them to
escape normal growth controls.
14.6/DNA Repair
Despite the high fidelity of DNA, replication errors still occur. With no way to correct errors,
cells would accumulate an unacceptable level of deleterious or lethal mutation. A balance must
exist between new variation arising by mutation, and the effects of deleterious mutations on
the individual.
Cells are constantly exposed to DNA-damaging agents
In addition to errors in DNA replication, cells are constantly exposed to DNA-damaging agents
such as radiation (UV light and X-rays) and chemicals in the environment. Agents that damage
DNA can lead to mutations, and any agent that increases the number of mutations above
background levels is considered a mutagen. Sunlight itself includes UV radiation and is thus
mutagenic. Ozone normally screens out much of the UV radiation in sunlight, but some
remains. Regions in the southern hemisphere show increases in skin cancer that correlate with
a seasonal “ozone hole”, indicating a relationship between sunlight and mutations.
We are also exposed to mutagens in our diet, as either contaminant or natural plant products
that are mutagenic. When a simple test to detect mutagens was devised, screening of possible
sources indicated an amazing diversity of mutagens in the environment and in natural sources.
As a result, consumer products are now screened to reduce the load of mutagens we are
exposed to, but we cannot escape natural sources.
DNA repair mechanisms
- Specific – targeting a particular type of DNA damage photo repair of thymine dimers
- Non-specific – able to repair any different kinds of DNA damage using a single
mechanism excision repair to correct damaged or mismatched nitrogenous base
Photo repair: when DNA is exposed to UV light, a number of photochemical reactions can
occur. The most common produces a thymine dimer, where adjacent thymines become
covalently linked together. There is a specific repair pathway, called photo repair, that
recognized this damage and reverses it.
Excision repair: a common form of nonspecific repair is excision repair. In this pathway, a
damage region is removed, or excised, and is then replaced by DNA synthesis. In E. coli, this
action is accomplished by proteins encoded by the uvr A, B, and C genes. Although these genes
were identified based on alleles that increased sensitivity of the cell to UV light (hence the “uvr”
in their names), the system can act on damage due to other mutagens.