Nucleus: DNA and DNA Synthesis
The familiar double membrane called the nuclear envelope surrounds the fluid material of the
nucleus. This membrane is often continuous with the endoplasmic reticulum. The nuclear
membrane has numerous pore-like structures that can be observed.
These pores account for as much as 25% of the nuclear membrane in some cells. The nuclear
membrane is known to be highly selective in what it permits to pass in and out of the nucleus. We
know that large mRNA molecules are assembled in the nuclear fluid and move into the cytoplasm.
Yet smaller molecules are not allowed to pass through the membrane.
The only obvious feature of the nuclear fluid (nucleoplasm) is the nucleolus (pl. nucleoli), which is
a structureless dense mass. It is the site of rRNA reproduction and appears to be the source of
ribosomes. As stated before, the nucleolus is made up of a collection of chromosomal loops.
Most importantly, the DNA, chromatin and chromosomes can be found inside the nucleus.
Chromatin is DNA that is combined with proteins, including histone and non-histone proteins.
When the cells are dividing, the chromatin is coiled into larger, highly visible bodies that are called
chromosomes. When the chromosomes 'relax' and diffuse, they are long and thread-like. Human
chromosomes per cell measure approximately 12 cm long end to end. Diffuse chromosomes
occupy the cell most of the time.
DNA is bunched up as tiny bead-like globules, each consisting of about 200 DNA base pairs wound
around the cluster consisting of 8 molecules of proteins called histones. Between each of these
bead-like globules are 50 DNA base pairs (open space).
Each chromosome consists of one long DNA molecule wrapped around globules of histones and
non-histone proteins.
Chromatin occurs in one of two forms: euchromatin or heterochromatin. Euchromatin contains
nearly all the functional genes. These are genes that we have been able to map and identify.
Heterochromatin contains the DNA that hasn't been identified as genes.
DNA Information and DNA Synthesis:
DNA stands for deoxyribonucleic acid. All cells need a set of instructions in order to survive. The
instructions are provided in the form of DNA. The structure of DNA was discovered in April of
1953 by James Watson and Francis Crick.
The "deoxy" implies that there is one less oxygen atom in each unit of DNA than RNA. The DNA
molecule can be broken down into three parts:
1) Nitrogen bases
2) Sugar backbone
3) Phosphate groups
The nitrogen bases are broken down into two groups: Purines and Pyrimidines.
Pyrimidines: single ring bases: cytosine and thymine.
Purines: double ring bases: guanine and adenine.
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The basic unit of DNA is a deoxynucleotide. These nucleotides can exist as free-floating
triphosphates in the cell fluids. When they're strung together, they form DNA strands.
In DNA synthesis, the bonds between two of the phosphates are broken to form strong covalent
bonds between nucleotides. In this process the two phosphates are freed leaving one phosphate
attached to the sugar. This phosphate will link two sugars together. Energy is released in the
breaking of these bonds, which allows for the joining of the molecules.
Eventually a long chain of nucleotides is produced. The backbone of the chain consists of
alternating sugar and phosphates linked by shared oxygen atoms. The nitrogen bases are side groups
of the sugar and are not part of the backbone.
The nucleotide chain has two ends. The end with the phosphate attached to the #5 carbon of the
sugar molecule is known as the 5' end. The end with the OH group attached to the #3 carbon of the
sugar molecule is called the 3' end.
In most cases, the nucleic acid chains are pictured with the 5' end on the left and the 3' end on the
right. Synthesis of the nucleic acid chain always proceeds from the 5' to the 3' end. The 5’
phosphate is added to the 3’ hydroxyl group.
DNA is almost always found in a double strand (due to the one less oxygen), while RNA is almost
always a single strand. Each strand of DNA is forged of strong covalent bonds, but H bonds only
weakly attract the two strands to each other. The configuration of the two strands of DNA wound
around each other is a double helix. As the sugar and phosphate are wound around each other, the
nitrogen bases point inwards. It looks as if someone had twisted a ladder (not down the middle but
like a spiral staircase) with the base pairs as the rungs. Following this image, we can see that the
sugar and phosphate groups make up the backbone or the sides of the ladder.
We see that Adenine will always form two hydrogen bonds with Thymine and that Guanine will
always form three hydrogen bonds with Cytosine.
The strands are antiparallel with sugar and phosphate backbones are going in opposite directions, ie.
One strand runs in the 5' to 3' direction and is paired with the second strand, which runs 3' to 5'.
The geometry of the base pairs is very precise. The A:T, T:A, C:G, and G:C pairs lie perfectly flat,
one pair on top of the next, like a stack of pennies.
Conditions that hold the DNA together are as follows:
1) Hydrogen Bonding: between base pairs.
2) Hydrophobic Interactions: DNA bases are insoluble in water, so they stick together in aqueous
solutions.
3) Base Stacking: When the bases lie on top of each other, base stacking interactions result.
Conditions for denaturing DNA.
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The hydrogen bonds may be broken in three ways:
a) Decrease the pH. H+ disrupt the hydrogen bonds by bonding to the slightly negative
atoms.
b) Increase the heat. The heat disrupts the hydrogen bonds by vibrating the Hydrogen
atoms.
c) Free Radicals: the extra electron on these molecules will break the hydrogen bonds by
bonding to the slightly positive hydrogen atom.
DNA Replication and Synthesis:
DNA synthesis occurs in a 5' to 3' direction. First an unwinding protein unwinds the two strands of
DNA, so that there is a region, a few hundred nucleotides long, in which two strands are unpaired
with their nucleotides sticking out into the cell fluid. Helicase is the enzyme responsible for the
separation of DNA strands. Once the strands are separated, a replication fork is established. In
prokaryotic cells, the DNA is in a circular form. In prokaryotes, DNA replication begins at the
Origins of Replication (Ori C). As the helicase unwinds a portion of the DNA, the other portion of
the molecule is stressed, due to the increased winding of the circular molecule. In order to relieve
this stress, an enzyme called topoisomerase breaks and reattaches the DNA molecule. The
unwinding of the DNA strand forms a replication fork.
An enzyme, DNA polymerase, begins to match the free-floating deoxynucleotides found in the cell
fluid to the bases presented by the opening up of the DNA strand. As nucleotides of the unwound
segment finds new partners, helicase unwinds more of the DNA strand creating a moving
replication fork.
Actually DNA Polymerase can only add a nucleotide to an already existing polynucleotide (chain of
nucleotides), which is paired with the complementary DNA strand. DNA polymerase cannot
initiate DNA synthesis, it can only add to the nucleotide chain. There is a pre-existing chain called
a PRIMER (a short RNA chain), which is placed on the DNA by PRIMASE. The primer is about
ten nucleotides long (in euks). As DNA synthesis occurs, another DNA polymerase replaces the
RNA nucleotides of the primer with DNA.
Lagging Strand Synthesis:
If we say that DNA synthesis occurs bidirectionally, then we can see that if one strand is put
together in a 5' to 3' direction, then the other part of the same strand must be put together in a 3' to 5'
direction. This is not precisely true. The strand that will be synthesized in an overall 3' to 5'
direction is called the lagging strand.
The lagging strand is made up of OKAZAKI FRAGMENTS, which are 1,000 to 2,000 base pairs in
length and synthesized in a 5' to 3' direction. Lagging strand DNA synthesis starts at a RNA
Primer, which is a sequence of approximately 10 nucleotides of RNA. The RNA primer is placed
down by an enzyme call Primase. The DNA polymerase lays down bases from the preprimary
complex in a 5' to 3' direction until DNA polymerase hits another RNA primer. Once the DNA
polymerase hits the primer, it stops laying bases and DNA bases replace the primer. Then DNA
Ligase seals up the newly laid DNA bases and Gyrase winds the DNA molecules up.
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Review of enzymes of DNA synthesis:
Helicase: unwinds
Primase: puts down RNA primer
DNA Polymerase: lays down the bases
Ligase: seals the bases up.
Gyrase: winds the DNA molecules.
Topoisomerase: relieves the tension in the replication fork.
Energetics of DNA Replication:
The nucleotides for DNA synthesis are assembled as triphosphates: deoxyadenosine triphosphate
(dATP), deoxyguanosine triphosphate (dGTP), deoxycytosine triphosphate (dCTP), and
deoxythymosine triphosphate (dTTP). The energy from the bonds between the phosphates provide
the energy to power the reactions catalyzed by DNA polymerase.
After the two phosphates have been removed, another enzyme breaks apart the two phosphates
recently cleaved from the triphosphate. These phosphates are now released as inorganic
phosphates.
The energy released allows the synthesis of DNA to be highly exergonic which prevents the
reaction from going in the reverse direction.
DNA synthesis is semi-conservative. Each new double helix is made up of one new strand and one
old strand. DNA synthesis is very accurate. DNA polymerase makes very few errors. But what
happens if there is a mistake in DNA synthesis? How are mistakes corrected?
DNA repair:
DNA polymerase not only puts bases down in order, but it also proofreads what it has laid down. If
the nucleotide that is laid down is the wrong one, polymerase will undo what it has done by going
back and removing the wrong base. However, DNA polymerase removes 10% of all bases put
down even if the base is correct. If the polymerase continues two bases past the mismatch, the
probability of DNA polymerase going back two bases to get the mismatch is very low. In a 5' to 3'
direction DNA polymerase lays down the base, but in the 3' to 5' direction it proof reads.
Photoreactivating Enzyme:
U.V. light is the easiest mutagen to work with. With U.V. light the most common lesion that
occurs and is called the pyrimidine-pyrimidine dimer or the thymine-thymine dimer. The T-T
dimer can be fixed by the photoreactivating enzyme (overhead).
T-T ------------------------------------> T T
Photoreactivating Enzyme
This enzyme is activated by the sunlight and breaks the thymine-to-thymine dimers. The thymineto-thymine dimers act as a deletion.
Excision Repair:
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Excision repair is responsible for repairing a broad spectrum of DNA damage ie. U.V. Damage and
Bulky Lesions. Bulky lesions are large molecules that attach to the DNA strand. These large
molecules will stop DNA synthesis.
The damages are all repaired by the excision repair system. Excision repair is able to
a) Repair compounds of different structures.
b) Make repairs when compounds bind in different places.
c) Detect damage in DNA.
There are three enzymes that make up the excision repair system:
Uvr A, Uvr B, and Uvr C.
Mechanisms for repair:
1) Recognition: some sort of recognition of damage. Enzymes bind in the vicinity of DNA lesion.
2) Incision: two nicks are made on either side of the lesion.
3) Displacement and resynthesis: Lesion is removed and DNA is resynthesized. This requires the
use of other enzymes (polymerase).
4) Seal the new DNA: Ligase does this.
Excision repair requires the presence of the complimentary strand of double helix during repair.
Complimenting the bases does repair.
We've talked about three types of DNA repair systems:
1) DNA polymerase: It is the proofreader in a 3'-5' direction.
2) Photoreactivating Enzyme: usually fixes T-T dimer.
3) Excision Repair System: Uvr A, B, C fixes bulky lesions.
When the repair system lets a lesion, mismatch or other mistake in the DNA synthesis occur, there
is a mutation.
Mutation: Inheritable change in DNA molecule.
These changes may result from errors in replication, from damage that repair enzymes do not
correct, or from spontaneous rearrangements in the DNA molecule.
A change in one or two nucleotides may have no effect on a cell or may be disastrous for the cell.
Mutations occur from mutagenic agents, mutagens such as radiation (X-ray and U.V. light).
Certain chemicals can cause a change in DNA.
Mutations produce variations in genetic material. Genetic variations are the raw material for
evolution. These mutations must occur in the gametes. Mutations in somatic (body cells) can lead
to cancer.
There are different types of mutations:
1) Change of one nucleotide to another. These are called mismatch or base substitutions. Caused
by mistakes in DNA polymerase.
*
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GATTCCTTAA ------------> GAATCCTTAA
2) Insertion of one or more nucleotides into the DNA sequence.
* *
GATTCCTTAA ------------> GAATTCCCTTAA
3) Deletion of one or more nucleotides from a DNA sequence. Can be caused by T-T dimers.
* **
GATTCCTTAA -----------> GA TT C TAA
4) Inversion of part of a nucleotide sequence.
* *
GATTCCTTAA -------> GATTCCAATT
5) Breakage and loss of a fragment of DNA. Can be caused by bulky lesions.
*
GATTCCTTAA ------------> GATTCCT
6) Extra copies of DNA.
* *
GATTCCTTAA -----------> GATTCCTTCCTTAA
GAC repeats are called polyglutamine diseases. The following genetic anomalies are caused by
GAC repeats: Huntington Chorea, Fragile X syndrome, and Alzheimers.
The ends of the DNA molecule:
Since DNA Polymerase can only add to the 3’ end of a pre-existing polynucleotide, this creates a
problem. There is no way to complete the 5’ end of the daughter DNA, because there is no DNA to
complement the primer to.
Here is an example of DNA synthesis:
5’----------------------------------------------------3’
3’----------------------------------------------------5’
5’----------------------------------------------------3’
3’
------------|---P-------5’*
5’
----P-------|-|----P------|3’
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3’----------------------------------------------------5’
*The primer is removed, but DNA polymerase needs another primer before the end, to add to it.
There is no DNA on the 3’end, so there can be no primer.
The DNA molecule gets shorter and shorter. In prokaryotes this is no problem because DNA is
circular (nucleoid). In euks, they have special ends of chromosomes called TELOMERES.
Telomeres are repeated nucleotide sequences about 7,000 to 10,000 bases long at birth (for
example, TTAGGG). These telomeres protect against the erosion of DNA through successive
DNA replication (these act as aglets). Telomerase is an enzyme that increases the length of
telomeres.
Telomerase has an RNA template for new telomeres. Telomerase is not present in most cells of
multicellular organisms. It is possible that telomeres limits our cells life span, because once the
telomeres are gone, the cell will die. Telomerase is present in germ cells, which gives rise to
gametes. The telomeres in gametes and newborns are very long.
There is evidence of Telomerase in cancer cells. Telomerase stabilizes the telomeres of some
cancer cells, which makes the cancer cells immortal. These cells can divide without loss of
telomeres.
TEP gene on chromosome 14 produces Telomerase. When you reach the age of 85, the telomeres
are 5/8 of their original length. Telomerase contains RNA that acts as a template to create the
telomeres (this resembles reverse transcriptase). All mammals, most animals, protists, fungi, plants
have TTAGGG as the telomere sequence.
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