Lecture 3 (2015)
Apunte InglésUniversidad | Universidad Internacional de Cataluña (UIC) |
Grado | Medicina - 1º curso |
Asignatura | Biologia Molecular |
Año del apunte | 2015 |
Páginas | 6 |
Fecha de subida | 10/03/2015 |
Descargas | 69 |
Subido por | iguarromarzoa |
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2014/2015
Molecular Biology
Ingrid Guarro Marzoa
Lecture 3: Replication and the DNA copier machine
•
Introduction:
Whatever replicates DNA or the nucleic acid, has to separate de two strands
and copy. The researchers that discovered the structure of DNA, as soon as
they saw it, knew that the structure had the answer of how DNA was replicated
just by assuming the information in one strand is enough to copy the other.
DNA-polymerases complete the new strand with the information of the old
strand. The process is quite easy, you only have to copy the old bases with
complementary bases and close the gaps with phosphodiester bonds.
•
Models of replication:
During replication, the semiconservative model proposed that each of the two
original DNA strands would serve as a template for the formation of an entire
new strand. However, scientist discussed different models of replication:
o Conservative model:
It was supposed that all the information on the parental DNA was
transmitted to the one of the subsidiary DNA helices.
o Semiconservative model:
They thought that each of the two daughters of a dividing cell inherits
a new DNA double helix containing one original and one new strand.
To prove this, analyses carried out in the early 1960s an experiment
that gave the answer. Experiment:
The cells that had the DNA at the beginning (parental DNA) had been
grown in a media with C14, an isotope of carbon different form the
natural, (it can be either nitrogen isotopes or carbon isotopes). They
considered that the DNA that contained a heavy isotope was “heavy”
so if that DNA was synthesized in the subsidiary generations, it will be
“heavy”. Then the subsidiary DNA was transferred to a media without
the heavy isotope, and was considered “light”. Thus, newly
synthesized DNA would be light. The analyses purified the DNA
samples and placed them in a gradient of density. The result showed
that the subsidiary DNA was lighter than the parental DNA. This
meant that DNA replication could not follow the conservative model.
They repeated the experiment and saw that the subsidiary DNA
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contained both heavy and light molecules, which supports the
semiconservative model.
•
Replication forks:
Replication forks are places of the DNA were nucleotides are replicated.
As the DNA presents an antiparallel structure, when it has to be replicated two
forks of replication are created. These two forks move in opposite directions (left
and right) and create what is known as replication eye.
During DNA replication inside a cell, each of the two original DNA strands
serves as a template for the formation of an entire new strand.
Depending on the organisms we can find:
a. Bidirectional replication: two forks replicate DNA.
b. Unidirectional replication: one fork replicates DNA. We find this
especially in virus. Right now, it’s being investigated the process of
replication in viruses to find molecules against viral replication.
There are different replication origins because DNA is very long and if not it
would be impossible to replicate DNA. When two forks collide, they create a
fused replicon. These origins are not all activated simultaneously. A replicon is
the distance between two replication origins: the DNA synthesized from one
single origin. This distance corresponds to the average distance between
origins. The distance between origins in our genome is about 200 thousand
base pairs. The size of our genome is 3 Gigabase pair. That means that our
cells have more that 10 thousand different origins per cell.
DNA combing: you purify DNA form cells and in solution you leave it there on a
glass surface but you do it on a way where DNA molecules lay as hair after
combing, creating very straight strands.
o Experiment:
Researchers couldn’t understand how can a DNA strand grow in
opposite directions so they carried out an experiment. They added a
chemically modified nucleotide to a cell. An antibody detects this
molecule (green segments in image) and like this you can detect the
origins of replication.
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Molecular Biology
Ingrid Guarro Marzoa
How are nucleotides added to the nascent strand?
During the replication process, we have to distinguish between two strands:
a. Template strands: the strand that is going to be copied
b. Nascent strand: the one that is going to be born
(new one).
Nucleotides are always added to the 3’ end. This is a universal rule. The energy
to create new bonds comes from the 5’ PPP (triple phosphate bond) of the
nucleotide being incorporated. The bond between phosphates is very unstable
and may break spontaneously. If nucleotides were added to the 5’ PPP ends,
and the triple phosphate bond breaks, we would not be able to continue with
replication (the new phosphodiester bond between nucleotides will not be
possible) so that’s why nucleotides provide the 5’ PPP end and are added in the
3’ end, because it’s more stable.
o DNA polymerases:
DNA polymerases have to:
a. Break the triple phosphate bond
b. Create a new phosphodiester bond
c. Check the complementarity: this enzyme checks if the new
base is complementary to the base in the parental strand. To
do this, it checks the angles and distances between the
nitrogens of the riboses. The active center (cavity that fits
perfectly the molecules that are going to react) of the DNA
polymerase has to fit perfectly with the base at the parental
strand, with the new nucleotide that enters to the new strand. If
this nucleotide can’t do the hydrogen bonds with the other
nucleotide, it can’t fit and the reaction is not done.
This reaction can also make mistakes. The rate is that 1 in a
million nucleotides there’s a mistake (we have 3 Gigabase
pairs, so everytime we replicate our DNA we would have about
3 thousand mutations. If that was the real error of mistakes, we
would die of cancer before birth.
d. Correct errors: If the base that enters to the DNA polymerase
is not the one that fits, the enzyme stays there and it breaks
the link and starts again.
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Molecular Biology
Ingrid Guarro Marzoa
DNA is antiparallel which means that DNA polymerase only could add
nucleotides in the strand that grows 5’à3’. This strand is the leading
strand because it’s the one that has the 3’ in de same direction as the
fork is moving. To replicate the other strand fragments are added in a
discontinued way. These fragments are called Okazaki fragments and
are that has between 1000-2000 base pairs. This strand is called the
lagging strand.
o Replication of the lagging strand
1. The primase adds a primer: primase creates a little primer of RNA. It
is a RNA primer because during the replication of the lagging strand,
we have to start from 0 and to start replication the primase (a RNA
polymerase) doesn’t need a nucleotide to start the process while DNA
polymerase always needs a nucleotide. Only RNA polymerases can
place the first one nucleotide.
2. Once we have the primer, primase goes away.
3. DNA polymerase checks if the primer is correctàstart of replication.
The lagging stands replicates until it finds the previous Okazaki fragment.
Once we have replicated the lagging strand, another DNA polymerase
degrades the RNA primer and synthesizes DNA instead, getting rid of possible
mutations. When this is done, a ligase nails the fragments.
o Other enzymes:
a. Helicases: go in front of the fork and they split the double helix.
They use ATP.
b. SSB: they protect the DNA until it’s replicated.
The replication process in both strands has to be coordinated. In order to make
this process efficient, both enzymes (DNA polymerases) are linked together
forming a dimer. With this junction, we have a strong complex that doesn’t allow
the two enzymes to go away.
The two enzymes synthesize DNA in different directions in space so to correct
this, the DNA polymerase that will produce the lagging strand is forced to make
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a loop (llaç, lazo). Replisomes (that are all proteins acting in the replication fork)
create this loop to synthesize both strands in the same spatial direction.
•
Initiation of replication at origins:
The first event after strand separation is performed by the primase, which
synthesizes a primer that will lead elongation of the leading strand in each
replication fork. Lagging strands will be replicated in a discontinuous manner as
Okazaki fragments as explained before. Orgins of replication are determined
by DNA sequence and a complex of proteins called ORC. As we will see next
year in Cell Biology, other proteins perform important roles to coordinate
replication with the cell cycle.
•
The end of replication:
While the leading strand has de DNA polymerase adding nucleotides until it
reaches the very end of the parental strand, the lagging strand will always have
a region of the parental strand that it’s not going to be replicated. The only
solution would be that the primase added a primer right at the end of the
parental chain but this enzyme can’t produce the RNA primer needed to start
the last Okazaki fragment at the very tip of a linear DNA molecule. This means
that the DNA at the very end of the parental strand will not be replicated.
To ensure the chromosome integrity and that during replication there’s not a
lose of information and to replenish the sequence of DNA that can’t be
synthetized, we have an enzyme called telomerase.
•
Telomere and telomerase:
Bacteria solve the “end-replication problem” by having circular DNA molecules
as chromosomes and eukaryotes have specialized nucleotide sequences at the
ends of their chromosomes that are incorporated intro structures called
telomeres. Telomeres in humans and mice have the sequence AGGGTT as
repeat unit, which is GGGGTT in unicellular eukaryotic organisms. This
sequence is repeated on average roughly a thousand times at each telomere.
This sequence is recognized by telomerase. Then telomerase elongates the
lagging strand in the 5’à3’ direction using an RNA template that is a
component of the enzyme itself. The telomerase places the RNA template and
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its 3 first bases complement the 3 last bases of the parental strand. The
enzymatic portion of telomerase resembles other enzymes that synthesize DNA
using the RNA template. By doing this, the parental strand elongates to
compensate for possible losses cuased by replication of the lagging strand.
Cancer cells express high levels of telomerase and normal cells have low levels
of telomerase. Having no telomerase can involve problems in normal
proliferating cells too because the lagging strand looses information from DNA.
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