lecture3_REPLICATION AND THE DNA COPIER MACHINE (2015)Apunte Inglés
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Molecular biology – Lecture 3
REPLICATION AND THE DNA COPIER MACHINE
Whatever replicates DNA or the nucleic acid has to separate the 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.
2. MODELS OF REPLICATION There are different hypothesis of replication of the DNA: - Conservative model: it was supposed that all the information on the parental DNA was transmitted to one of the new helices 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 aril 1960s an experiment that gave the answer.
Experiment: The cells that had the DNA at the beginning (parental DNA) had been grown in a C14 milieu, an isotope of carbon different from the normal one; (it can be ether nitrogen isotopes or carbon). They considered that the DNA that contained a different isotope was heavier, so if the DNA was synthesized with it, would be heavier too. Then that DNA was transferred to a normal carbon milieu, and it 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 results showed that the subsidiary DNA was lighter than the parental DNA. This meant that the DNA replication could not follow the conservative model. The repeated the experiment and they saw that the subsidiary DNA contained both heavy and light molecules, which supports the semiconservative model.
Molecular biology – Lecture 3 3. REPLICATION FORKS Replication forks are places of the DNA where the replication starts. 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 direction and create what is known as replication eye.
During DNA replication inside a cell, each of the two original strands serves as a template for the formation of an entire new strand.
Depending on the organisms we can find: - Bidirectional replication: two forks replicate DNA Unidirectional: one fork replicates DNA. We find this especially in virus.
There are many 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 replication origins: the DNA synthesized from one single origin. This distance in our genome is about 200 thousand base pairs-. The size of our genome is 3 gigabase pair (3x10^9). That means that our cells have more than 10 thousand origins per cell.
The replication fork drives DNA synthesis in a semiconservative mode.
The replication fork is the region of DNA in which there is a transition from the unwound parental duplex to the newly replicated daughter duplexes.
Replicated DNA is seen as a replication eye flanked by nonreplicated DNA.
Molecular biology – Lecture 3 4. HOW ARE THE NUCLEOTIDES ADDED TO THE NASCENT STRAND? During the replication process, we have to distinguish between two strands: a. Template strands: that strand that is going to be copied b. Nascent strand: the one that is going to be born (the new one) DNA synthesis occurs by adding nucleotides to the 3’ OH end of the growing strand, so that the new strand is synthesized in the 5’-3’ direction. This is a universal rule.
The energy to create new bones comes from a triple phosphate bond of the nucleotide being incorporated, which loses the terminal two phosphates groups in the reaction, given that the bond between phosphates is very unstable and may break spontaneously. If nucleotides were added to the 5’ ends, and the triple phosphate bond breaks, we would not be able to continue with replication (the new phosphodiester bond between nucleotides won’t be possible) so that’s why nucleotides provide 5’ PPP end and are added in the 3’ end.
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 nitrogen of the ribose. 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 fir and the reaction is not done.
d. Correct errors: if the base that enters to the DNA polymerase is not the one that fits, the enzyme says there and it breaks the link and starts again DNA polymerases incorporates the wrong nucleotide 1 on a million times 10^-6. We have 3 gigabase pairs, so every time 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 DNA is antiparallel what means that DNA polymerase only add nucleotides in the strand that grows 5’ 3’. This strand is the leading strand because it’s the one that has the 3’ in the same direction as the fork moving. To replicate the other strand, fragments are added in a discontinued way. These fragments are called Okazaki fragments and they have between 10002000 base pairs. This strand is called the lagging strand.
Molecular biology – Lecture 3 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 primase doesn’t need a nucleotide to start the process while DNA polymerase always needs a nucleotide. Only RNA polymerase can place the first one nucleotide.
2. Once we have the primer, primase goes away 3. DNA polymerase checks if the primer is correct start of replication The lagging strands replicates until it founds the previous Okazaki fragment. Once we have replicated the lagging strand, another NDA polymerase degrades the RAN primer and synthesizes DNA instead, getting rid of possible mutations.
When this is done, a ligase nails the fragments Other enzymes: - Helicases: go in front of the fork and they split the double helix. They use ATP SSB (Single Stranded binding proteins): they protect NDA 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 these conjunctions, 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 a loop. Replisomes (all proteins in the replication fork) crate this loop to synthesize both strands in the same spatial direction.
Molecular biology – Lecture 3 5. 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 strand in each replication fork. Lagging strands will be replicated in a discontinuous manner as Okazaki fragments as explained before. Origins of replication are determined by DNA sequence and a complex of proteins called ORC. There are other proteins that perform important roles to coordinate replication with the cell cycle.
6. THE END OF REPLICATION While the leading strand has the NDA polymerase adding nucleotides until it reaches the very end of the paternal strand the lagging strand will always have a region of the paternal strand that it’s not going to be replicated. The only solution that the primase added a primer at the very end of the paternal strand, but this enzyme can’t produce the RNA primer needed to start the last Okazaki fragment. This means that the DNA at the very end of the paternal strand won’t be replicated.
To ensure the chromosome integrity and that during replication there’s not a loss of information and to replenish the sequence of DNA that can’t be synthesized; we have an enzyme called telomerase.
7. TELOMERE AND TELOMERASE Bacteria solve the “end-replication” problem by having circular DNA molecules as chromosomes and eukaryotes have specialized nucleotide sequences at the end of their chromosomes that are incorporated into structures called telomeres. Telomeres in humans and mice have the sequence AGGGTT as a repeated unit, which is GGGGTT in unicellular eukaryotic organisms. This sequence is repeated on average roughly a thousand times at each telomere.
Molecular biology – Lecture 3 This sequence is recognized by telomerase. Then telomerase elongates the lagging strand in the 5’3’ direction using RNA template that is a component of the enzyme itself. The telomerase places the RNA template its 3 first bases complement the 3 last bases of the paternal strand. The enzymatic portion of telomerase resembles other enzymes that synthesize DNA using the RNA template. By doing this, the paternal strand elongates to compensate for possible losses caused 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 loses information from DNA.