DNa replication |
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Within the human body, billions of new cells are created every day. In order to meet this demand and ensure that cells have all the information they need to function properly, the DNA contained in the nucleus of these cells must be copied. This copying process is known as DNA replication and occurs within the nucleus of dividing cells. Normally copying all of the genetic information within a cell would take more than 95 years. However, thanks to the efficiency of the molecular machinery within cells, it can be accomplished in 6 to 8 hours.
Semiconservative replication
But how does the DNA create an identical copy of itself to ensure the daughter cell contains the exact same genetic information? It does this by using the original DNA strands as templates in what’s known as semiconservative replication. In semiconservative replication the double stranded molecule of DNA unzips to expose individual base pairs. These exposed bases can then be used to add new complementary base pairs creating two new daughter strands of DNA. This means that after one round of replication, each daughter strand contains one half of the original parent and one half new DNA.
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This process of semiconservative replication was identified by Matthew Meselson and Franklin Stahl through experimentation with E. coli. Their experiments involved exposing E. coli to different isotopes of nitrogen since DNA is nitrogenous based. Initially the E. coli was cultured in a medium of 15N making its entire DNA 15N. After culturing the bacteria in this medium, it was centrifuged to visualize a 15N band. The E. coli was then transferred to a 14N medium for a single generation. What they found was that when the bacteria were centrifuged again, it did not match the 15N band but showed an intermediate band. This was because the original 15N strands of DNA were used as the template strand with the new strand being synthesized from the mediums 14N. When a second generation was cultured and centrifuged, the intermediate band remained as the 15N DNA was still being used as a template but an even lighter, completely 14N band formed as well since the “new” 14N strands from the first generation also acted as templates for the second generation. |
Steps of DNA replication
The actual process of DNA replication can be broken down into a number of key steps:
1. DNA gyrase relaxes double helix as it unwinds
2. DNA helicase unzips the DNA strands
3. Single-stranded binding proteins binds to exposed DNA strands
4. Primase creates RNA primers used to start strand synthesis
5. DNA polymerase III synthesizes new complementary strand
6. DNA polymerase I replaces RNA with DNA
7. DNA ligase joins remaining gaps
8. DNA polymerase I and III check for and correct errors
1. DNA gyrase relaxes double helix as it unwinds
2. DNA helicase unzips the DNA strands
3. Single-stranded binding proteins binds to exposed DNA strands
4. Primase creates RNA primers used to start strand synthesis
5. DNA polymerase III synthesizes new complementary strand
6. DNA polymerase I replaces RNA with DNA
7. DNA ligase joins remaining gaps
8. DNA polymerase I and III check for and correct errors
Unzipping the dna
In order for DNA to be read and replicated it must first be unwound and separated into individual strands. This is accomplished by the enzyme DNA helicase. DNA helicase breaks the hydrogen bonds between the complementary base pairs. But what prevents the strands from reforming these bonds? Once the bonds are broken single-stranded binding proteins (SSBs) join onto the bases to prevent bond reformation. The unwinding and untwisting of DNA also causes tension to form in the molecule. This is similar to what happens if two strings were twisted together and then quickly pulled apart. DNA gyrase relieves this tension during this stage of replication.
As the DNA is unzipped, replication can begin. It begins at what are known as origins of replication. These origins of replication become replication bubbles. This is where the efficiency of eukaryotic DNA replication shines and allows for the 6 to 8 hour replication time. In eukaryotic cells, there are multiple replication bubbles, allowing replication to begin at multiple sites, simultaneously. At each replication bubble there are two replication forks that move in opposite directions.
Building the Complementary Strands
The new complementary strands of DNA are built by DNA polymerase III. However, DNA polymerase III needs a guide to know where to begin adding on complementary bases. The enzyme primase fills this role by attaching complementary RNA primers on the exposed template strand. DNA polymerase III uses these primers and begins synthesizing new DNA from the 5’ to 3’ direction. An extremely important distinction to make is that DNA polymerase III reads the template strand from 3’ to 5’ but the new strand is synthesized from 5’ to 3’. This is known as the directionality of DNA polymerase III.
As a result of this directionality, one strand of DNA is synthesized continuously and the other discontinuously. The continuous strand is known as the leading strand and is created in the direction of the replication fork. The discontinuous strand is called the lagging strand and is synthesized in away from the replication fork. The lagging strand is also synthesized in short segments called Okazaki fragments, named for Reiji Okazaki. Each of these fragments begins with an RNA primer to allow DNA polymerase III to find each fragment. |
Following the new complementary strand synthesis, another DNA polymerase enzyme, DNA polymerase I goes along the new strand and removes the RNA primers. After removing the primers it replaces them with DNA bases. A final enzyme called DNA ligase then joins the resulting gaps in the phosphate sugar backbone that remain from replacing all of the RNA primers. This is the last step of actual replication and results in new and complete strands of DNA.
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Quality control and repair
Throughout DNA replication, as the new DNA strand is being synthesized, both DNA polymerase I and III are also fulfilling a quality control role. If any base pairs have been improperly matched, these two enzymes can act as an exonuclease to cut out and correct the mistake before more of the strand is created. This is a crucial step, with repairs needing to occur immediately, to prevent the DNA from being filled with mistakes that could cause harm to the cell or organism.
dna replication summary
An animation that summarizes the process of DNA replication.
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Another, realistic, summary animation of DNA replication.
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