What is DNA Polymerase

DNA polymerase is an enzyme that catalyzes the polymerization of deoxyribonucleotides into a DNA strand. DNA polymerases are best-known for their role in DNA replication, in which the polymerase “reads” an intact DNA strand as a template and uses it to synthesize the new strand.

This process copies a piece of DNA. The newly-polymerized molecule is complementary to the template strand and identical to the template’s original partner strand. DNA polymerases use a magnesium ion for catalytic activity.

DNA polymerases have highly-conserved structure, which means that their overall catalytic subunits vary, on a whole, very little from species to species. Conserved structures usually indicate important, irreplicable functions of the cell, the maintenance of which provides evolutionary advantages.

A surface representation of human DNA polymerase β (Pol β), a central enzyme in the base excision repair (BER) pathway. Image Credit: niehs.nih.gov

Some viruses also encode special DNA polymerases, such as Hepatitis B virus DNA polymerase. These may selectively replicate viral DNA through a variety of mechanisms. Retroviruses encode an unusual DNA polymerase called reverse transcriptase, which is an RNA-dependent DNA polymerase (RdDp). It polymerizes DNA from a template of RNA.

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DNA Evolution

DNA contains the genetic information that allows all modern living things to function, grow and reproduce. However, it is unclear how long in the 4-billion-year history of life DNA has performed this function, as it has been proposed that the earliest forms of life may have used RNA as their genetic material. RNA may have acted as the central part of early cell metabolism as it can both transmit genetic information and carry out catalysis as part of ribozymes.

This ancient RNA world where nucleic acid would have been used for both catalysis and genetics may have influenced the evolution of the current genetic code based on four nucleotide bases. This would occur since the number of different bases in such an organism is a trade-off between a small number of bases increasing replication accuracy and a large number of bases increasing the catalytic efficiency of ribozymes.

Unfortunately, there is no direct evidence of ancient genetic systems, as recovery of DNA from most fossils is impossible. This is because DNA will survive in the environment for less than one million years and slowly degrades into short fragments in solution. Claims for older DNA have been made, most notably a report of the isolation of a viable bacterium from a salt crystal 250 million years old, but these claims are controversial.

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DNA Polymerase Function

DNA polymerase function is can add free nucleotides to only the 3′ end of the newly-forming strand. This results in elongation of the new strand in a 5′-3′ direction. No known DNA polymerase is able to begin a new chain (de novo). DNA polymerase can add a nucleotide onto only a preexisting 3′-OH group, and, therefore, needs a primer at which it can add the first nucleotide. Primers consist of RNA and/or DNA bases. In DNA replication, the first two bases are always RNA, and are synthesized by another enzyme called primase. An enzyme known as a helicase is required to unwind DNA from a double-strand structure to a single-strand structure to facilitate replication of each strand consistent with the semiconservative model of DNA replication.

Error correction is a property of some, but not all, DNA polymerases. This process corrects mistakes in newly-synthesized DNA. When an incorrect base pair is recognized, DNA polymerase reverses its direction by one base pair of DNA. The 3′-5′ exonuclease activity of the enzyme allows the incorrect base pair to be excised (this activity is known as proofreading). Following base excision, the polymerase can re-insert the correct base and replication can continue.

Various DNA polymerases are extensively used in molecular biology experiments.

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DNA Polymerase

DNA polymerases are a family of enzymes that carry out all forms of DNA replication. However, a DNA polymerase can only extend an existing DNA strand paired with a template strand; it cannot begin the synthesis of a new strand. To begin synthesis, a short fragment of DNA or RNA, called a primer, must be created and paired with the template DNA strand.

DNA polymerase then synthesizes a new strand of DNA by extending the 3′ end of an existing nucleotide chain, adding new nucleotides matched to the template strand one at a time via the creation of phosphodiester bonds. The energy for this process of DNA polymerization comes from two of the three total phosphates attached to each unincorporated base. (Free bases with their attached phosphate groups are called nucleoside triphosphates.) When a nucleotide is being added to a growing DNA strand, two of the phosphates are removed and the energy produced creates a phosphodiester bond that attaches the remaining phosphate to the growing chain. The energetics of this process also help explain the directionality of synthesis – if DNA were synthesized in the 3′ to 5′ direction, the energy for the process would come from the 5′ end of the growing strand rather than from free nucleotides.

In general, DNA polymerases are extremely accurate, making less than one mistake for every 107 nucleotides added. Even so, some DNA polymerases also have proofreading ability; they can remove nucleotides from the end of a strand in order to correct mismatched bases. If the 5′ nucleotide needs to be removed during proofreading, the triphosphate end is lost. Hence, the energy source that usually provides energy to add a new nucleotide is also lost.

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DNA Model

Deoxyribonucleic acid (DNA) is a molecule that contains all the information to determine who you are and what you look like.

The chemical compound that makes up DNA was first discovered by Friedrich Miescher in Germany around 1869. In 1953, Francis Crick and James Watson discovered that DNA is shaped like a ladder coiled into a ‘double helix’ shape.

The ‘sides’ of the ladder are a linked chain of alternating sugar and phosphate molecules.

The ‘rungs’ of the ladder are attached to the sugar molecules. Each rung is made up of two chemicals called bases. There are four
different bases – adenine (A), thymine (T), guanine (G) and cytosine (C) and they link together in pairs (A with T, C with G) to form a rung. The order of the bases and rungs creates a kind of code for the DNA information.

Your body is made up of many different chemicals. An important group of chemicals is the proteins, which build your body and help it to function.

Each protein is formed from over 100 amino acids. There are 20 different types of amino acids that can be used to make proteins.

The code in the DNA ladder’s rungs is a recipe for building proteins. Tiny particles called ribosomes follow the DNA recipe to bind amino acids together and build proteins. Up to 1 000 rungs might be needed to hold the recipe for just one protein.

A group of rungs that carries the recipe for one protein is called a gene. When many genes are linked together in a DNA ‘ladder’, they will form a chromosome.

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Function of DNA

DNA is found primarily in the nucleus of a cell in strands of genetic material called chromosomes. Each chromosome is a single piece of double stranded DNA; specific areas of the chromosome that are responsible for particular body functions are called genes.

The instructions coded by the 3-base sequences are actually carried to the areas in the cells where protein manufacture occurs by ribonucleic acid (RNA).

The structure (shape) and function of the thousands of proteins in an organism is controlled by the order of the amino acids in the protein, and therefore is ultimately controlled by the sequence of bases on the DNA. For example if one amino acid is substituted in the protein haemoglobin, the condition known as sickle cell anemia occurs, this condition is due to a single base change in the DNA that codes for this protein

When a cell divides the double stranded DNA is “unzipped”, and new DNA strands form using the single strands from the original DNA as templates thus replicating the sequence of DNA bases.

Many proteins and enzymes are involved in the process of DNA replication, one particular group the DNA polymerases are now used in the analysis of minute traces of DNA found at a crime scene as part of a technique known as POLYMERASE CHAIN REACTION (PCR).

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DNA Isolation

DNA isolation is a routine procedure to collect DNA for subsequent molecular or forensic analysis. There are three basic and one optional steps in a DNA extraction:

Breaking the cells open, commonly referred to as cell disruption or cell lysis, to expose the DNA within. This is commonly achieved by grinding or sonicating the sample.

Removing membrane lipids by adding a detergent.
Removing proteins by adding a protease (optional but almost always done).
Precipitating the DNA with an alcohol — usually ice-cold ethanol or isopropanol. Since DNA is insoluble in these alcohols, it will aggregate together, giving a pellet upon centrifugation. This step also removes alcohol-soluble salt.

Refinements of the technique include adding a chelating agent to sequester divalent cations such as Mg2+ and Ca2+, which prevents enzymes like DNAse from degrading the DNA.

Cellular and histone proteins bound to the DNA can be removed either by adding a protease or by having precipitated the proteins with sodium or ammonium acetate, or extracted them with a phenol-chloroform mixture prior to the DNA-precipitation.

If desired, the DNA can be resolubilized in a slightly alkaline buffer or in ultra-pure water.

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