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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DNA Methylation

DNA methylation is a crucial part of normal organismal development and cellular differentiation in higher organisms. DNA methylation stably alters the gene expression pattern in cells such that cells can “remember where they have been” or decrease gene expression; for example, cells programmed to be pancreatic islets during embryonic development remain pancreatic islets throughout the life of the organism without continuing signals telling them that they need to remain islets.

In addition, DNA methylation suppresses the expression of viral genes and other deleterious elements that have been incorporated into the genome of the host over time.

DNA methylation involves the addition of a methyl group to the 5 position of the cytosine pyrimidine ring or the number 6 nitrogen of the adenine purine ring (cytosine and adenine are two of the four bases of DNA).

This modification can be inherited through cell division. DNA methylation is typically removed during zygote formation and re-established through successive cell divisions during development although the latest research shows that hydroxylation of methyl group occurs rather than complete removal of methyl groups in zygote.

DNA methylation also forms the basis of chromatin structure, which enables cells to form the myriad characteristics necessary for multicellular life from a single immutable sequence of DNA.

DNA methylation also plays a crucial role in the development of nearly all types of cancer.

DNA methylation involves the addition of a methyl group to DNA – for example, to the number 5 carbon of the cytosine pyrimidine ring – in this case with the specific effect of reducing gene expression. DNA methylation at the 5 position of cytosine has been found in every vertebrate examined. In adult somatic tissues, DNA methylation typically occurs in a CpG dinucleotide context; non-CpG methylation is prevalent in embryonic stem cells.

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DNA Function

DNA usually occurs as linear chromosomes in eukaryotes, and circular chromosomes in prokaryotes. The set of chromosomes in a cell makes up its genome; the human genome has approximately 3 billion base pairs of DNA arranged into 46 chromosomes. The information carried by DNA is held in the sequence of pieces of DNA called genes. Transmission of genetic information in genes is achieved via complementary base pairing. For example, in transcription, when a cell uses the information in a gene, the DNA sequence is copied into a complementary RNA sequence through the attraction between the DNA and the correct RNA nucleotides. Usually, this RNA copy is then used to make a matching protein sequence in a process called translation, which depends on the same interaction between RNA nucleotides. In alternative fashion, a cell may simply copy its genetic information in a process called DNA replication. The details of these functions are covered in other articles; here we focus on the interactions between DNA and other molecules that mediate the function of the genome.

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DNA Structure

Illustration of the double helical structure of the DNA molecule.
The structure of DNA is illustrated by a right handed double helix, with about 10 nucleotide pairs per helical turn. Each spiral strand, composed of a sugar phosphate backbone and attached bases, is connected to a complementary strand by hydrogen bonding (non- covalent) between paired bases, adenine (A) with thymine (T) and guanine (G) with cytosine (C).
Adenine and thymine are connected by two hydrogen bonds (non-covalent) while guanine and cytosine are connected by three.
This structure was first described by James Watson and Francis Crick in 1953.

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DNA

Deoxyribo nucleic acid or DNA is a nucleic acid that contains the genetic instructions used in the development and functioning of all known living organisms (with the exception of RNA viruses). The main role of DNA molecules is the long-term storage of information. DNA is often compared to a set of blueprints, like a recipe or a code, since it contains the instructions needed to construct other components of cells, such as proteins and RNA molecules. The DNA segments that carry this genetic information are called genes, but other DNA sequences have structural purposes, or are involved in regulating the use of this genetic information. Along with RNA and proteins, DNA is one of the three major macromolecules that are essential for all known forms of life.
DNA consists of two long polymers of simple units called nucleotides, with backbones made of sugars and phosphate groups joined by ester bonds. These two strands run in opposite directions to each other and are therefore anti-parallel. Attached to each sugar is one of four types of molecules called nucleobases (informally, bases). It is the sequence of these four nucleobases along the backbone that encodes information. This information is read using the genetic code, which specifies the sequence of the amino acids within proteins. The code is read by copying stretches of DNA into the related nucleic acid RNA, in a process called transcription.
Within cells, DNA is organized into long structures called chromosomes. These chromosomes are duplicated before cells divide, in a process called DNA replication. Eukaryotic organisms (animals, plants, fungi, and protists) store most of their DNA inside the cell nucleus and some of their DNA in organelles, such as mitochondria or chloroplasts. In contrast, prokaryotes (bacteria and archaea) store their DNA only in the cytoplasm. Within the chromosomes, chromatin proteins such as histones compact and organize DNA. These compact structures guide the interactions between DNA and other proteins, helping control which parts of the DNA are transcribed.

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DNA and RNA

DNA and RNA are two different nucleic acids found in the cells of every living organism. Both have significant roles to play in cell biology. DNA and RNA structure are similar because they both consist of long chains of nucleotide units. However, there are a few structural details that distinguish them from each other, and if you are to compare DNA and RNA, these would be the results:
RNA is single-stranded while DNA is a double-stranded helix. RNA also has uracil as its base while the DNA base is thymine. However, even with the differences in their structures, DNA and RNA have cooperating roles in the field of Cell Biology.
DNA contains the genetic information of an organism, and this information dictates how the body’s cells would construct new proteins according to the genetic code of the organism. Within the cell structure, DNA is organized into structures called chromosomes, which are duplicated during cell division.
These chromosomes would then release the genetic codes that will be transcribed and carried by the RNA (specifically the messenger RNA) to the ribosome. The ribosome will then synthesize new proteins that will help the body grow. This is the how the DNA and RNA work together in the body.
Resource : science.plazza.us

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