Lecture 3 (2015)

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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 2014/2015 Molecular Biology Ingrid Guarro Marzoa 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.
2014/2015 • 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.
2014/2015 Molecular Biology Ingrid Guarro Marzoa DNA is antiparallel which means that DNA polymerase only could add nucleotides in the strand that grows 5’à3’. 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 correctàstart 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 2014/2015 Molecular Biology Ingrid Guarro Marzoa 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’à3’ direction using an RNA template that is a component of the enzyme itself. The telomerase places the RNA template and 2014/2015 Molecular Biology Ingrid Guarro Marzoa 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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