DNA replication

DNA Replication (also known as DNA synthesis) is a process where the double stranded Deoxyribonucleic Acid (DNA) is copied. Replication is an important first step in cell division, as cells must duplicate their entire genetic constitution before they can divide into two daughter cells. DNA replication is also a critical requirement for DNA repair.

Requirements of DNA Replication
Replication requires three important components in order for this process to work.

Template Strand
The DNA serves as a template to guide the incoming nucleotides. (Purves,220) The template strand attaches to the RNA primer strand in order to replicate the strand of DNA. The template just guides the nucleotides to the place that they need to go in. This is another component that is needed in order for DNA replication to even take place. If the DNA only had the template strand and none of the rest of the things then the strand could not replicate. The template strand is very thin. Through an enzyme called primase the DNA template strand is synthesized one nucleotide at a time. (Purves, 224)

DNA Polymerase
There are many DNA polymerases within a cell, but only one of which is used in the replication of DNA. In order for DNA to replicate, it must have a primer, which is a small strand of RNA. The DNA template strand synthesizes one nucleotide at a time by the use of primase. The adding of nucleotides to the 3’ end, which is continued until that section of DNA is replicated. The other DNA polymerases that are within the cell are used in other ways besides replication. DNA polymerase III is one certain polymerase, which aids in replication of what is known as the “lagging strand”. DNA ligase is the final enzyme, which combines the lagging strand.(Purves, 225-226)

Free 3' Hydroxyl
The free 3' hydroxyl is the starter strand called a primer which is required in order for DNA to be replicated. The primer strand is not very big, it is shorter then a single strand of RNA. The primer is the complementary strand to the template strand of DNA. This is another one of the things that are needed in order to make the process of DNA replication even possible. The DNA polymerase is added to the 3' end of the primer, which keeps growing until the section is finally complete. After a while the RNA primer is degraded and disappears.(Purves, 224)

Types of Replication
During the discovery of DNA replication they also discovered that there are three modes of replication that a DNA strand can take. The three types of DNA replication are semiconservative, conservative, and dispersive. These three types of replication were tested on in order to see the possible patterns that would result in the complementary base pairing. (Purves, 220)

Semiconservative Replication
Through this type of replication the parent strand serves as a template for the new strand. The two offspring would have one of the parent strand and one new strand. (Purves, 220)

Conservative Replication
Through this type of replication the double helix serves as a template. This although does not contribute to the new double helix. (Purves, 220)

Dispersive Replication
In this type of replication the fragments from the parent DNA molecule. The DNA serves as a template for the assembly of the new molecule. The new double helix contains old and new parts of the DNA strands. (Purves, 220-221)

DNA Repairing
After the DNA is replicated, there are three DNA repair mechanisms. They are to be used when they feel necessary. This will lessen the chance of human mutation. The rate of error in which the DNA polymerases makes a mistake is very high. This is why the DNA has mechanisms to cause fewer mutations. The three mechanisms are:

Proofreading
Every time a nucleotide is introduced to an already growing chain, the DNA polymerase checks the connection. If the pair is mismatched, it will be removed and redone. This lowers the overall rate of mutations. (Purves, 227)

Mismatch Pair
After the replication takes place, another set of proteins check to make sure that there are no more mismatched base pairs and also which strand is the wrong one.

Excision Pair
There can still be damage while the cell is living. Because of this, there are enzymes constantly checking the cell. If they detect a problem, the enzyme cuts the strand and also cuts away the opposite base. DNA polymerase and ligase then fix this base sequence.

Laboratory Technique
Scientists use two techniques in a laboratory that involve DNA replication. They use these techniques to observe genes and genomes. One of the techniques can make multiple copies of DNA from only a short piece. Another technique allows scientists to determine the base sequence of DNA. The two techniques are: (Purves, 228)

Polymerase chain reaction (PCR)
Through this process, a short strand of DNA is copied repetitively. There are three steps that are repeated in the process. The DNA strands are denatured or heated in order to separate the strand. Next, they add a primer, which was artificially made. They also add DNA polymerase and the four deoxyribonucleotide triphosphates. These three are needed in order to replicate DNA. The final step is then the DNA polymerase catalyzes the production of a complementary strand. (Purves, 229)

DNA Sequencing
This allows scientists to determines the base sequence of a DNA. The process is the DNA is first denatured and a single strand is placed in a test tube. They then add DNA polymerase, primer, the four dNTP’s, and small amounts of ddNTP’s. The DNA replicates within the tube and after the DNA fragments are denatured. The fragments then go through electrophoresis, which sorts the lengths of DNA fragments. The fragments then pass through a laser beam which excites the fluorescent tags. This information is fed into a computer at which tells the sequence. (Purves, 231)

Mendelson- Stahl Experiment
Through this experiment Meselson- Stahl convinced the scientists that the correct model for DNA replication was the semiconservative replication. They used density labeling in order to distinguish the old and new strands of DNA. In their experiment they used “heavy” isotopes of nitrogen. This nitrogen is a nonradioactive isotope that made the molecule more dense. The chemically identical molecules containing an isotope known as 14 N. In the first part of their experiment they grew cultures of Escherichia coli. One of the cultures was grown in 15N, which made the DNA “heavy”. The other culture was placed in a medium using 14N rather than 15N, which made all the DNA “light”. In the next part of their experiment they grew E. coli in the 15N medium and then transferred the bacteria from that medium to the 14N. Through this they came up with the conclusion that through E. coli DNA replicates every 20 minutes. They then took DNA samples from each generation through, which they found that the density gradient was different each generation. Their observations could only be explained with the semiconservative model of DNA replication. Their results showed that when the DNA first underwent replication, it was in 15N, which caused the DNA to be heavy. Because in the semiconservative model of DNA replication, the one strand acts as a template for a second strand which the DNA was in a 14N DNA strand and a 15N and they were of intermediate density.(Purves, 221 - 222)