DNA-Polymerase

• CAS Number: 9012-90-2

DNA polymerases, more DNA-dependent DNA polymerases are enzymes which catalyze the synthesis of DNA from a DNA template to deoxyribonucleotides. DNA polymerases have a key role in the DNA replication.

Biochemical aspects

Polymerase activity

The polymerase enables the chemical linkage of individual molecules (monomers ) to form a chain ( polymer). In the case of DNA polymerase, the polymer formed is the deoxyribonucleic acid ( DNA), are used as monomers, deoxyribonucleotides, more deoxy- nucleoside triphosphates (dNTPs ). The DNA polymerase uses always an existing single-strand DNA as a template ( template) for the synthesis of a new complementary strand, whose nucleotide sequence is thus determined by the die. This preservation of the DNA sequence is critical for the ability of DNA polymerase to copy the DNA encoded genetic information. The correct copy of the template is achieved by complementary base pairing of nucleotide bases built with the bases of the DNA template mediated by hydrogen bonds. The synthesis of the new DNA strand is carried out by the 5'- to the 3'- end. Chemically, this takes place is a nucleophilic attack of the 3'- terminal hydroxyl group of the DNA strand of the α - phosphate of the dNTP, wherein pyrophosphate is released. This step is catalyzed by the polymerase.

In contrast to RNA polymerases can only be the synthesis of the complementary DNA strand in DNA polymerases if the polymerase is a free 3' - hydroxy end available. To this then the first nucleotide is attached. The polymerase chain reaction ( PCR), one uses for this purpose an about 15-20 nucleotide long single-stranded DNA (primer ), which serves as a starting point of reaction. DNA polymerases generally require magnesium ions as a cofactor.

Catalyze the formation of the diester is carried out functionally similar to the corresponding reaction of the RNA polymerase. The last nucleotide of the already synthesized section and the nucleotide to be joined are coordinated to each one of two magnesium ions in the catalytic center of the polymerase domain. The first phosphate group of the nucleotide to be joined is coordinated to two magnesium ions. The spatial location allows an attack by the hydroxyl group of the previous nucleotide to the phosphate group of the to be joined. Here, a pyrophosphate is cleaved.

Exonuclease activity

Many polymerases also have other enzyme functions. To ensure that there are no errors in reading the DNA template, they have a so-called proofreading function (English proofreading ), ie they are able to detect this and then remove, from the DNA of the incorporation of a mismatched nucleotide. This ability is referred to as the 3 '→ 5' exonuclease activity. Some polymerases also possess a 5 '→ 3' exonuclease activity. This allows the degradation of an existing DNA or RNA strand, which is paired with the template strand, while a new strand is formed. This results in a replacement of the old string with a new string. This exonuclease activity is used in the method of nick translation.

Various DNA polymerases

In bacteria, such as Escherichia coli, there are three different polymerases. One of them, the DNA polymerase I ( Pol I) was isolated in 1955 by Arthur Kornberg and was the first polymerase at all, which was discovered. But this is not the most important for replication in E. coli polymerase, since it catalyzes only about 20 synthetic steps (Man she says has only a low processivity ). DNA polymerase II, DNA polymerase III and the other two DNA polymerases in E. coli were isolated 15 years after the discovery of the DNA polymerase I, after E. coli mutants with defects in the polymerase I gene as yet replication-competent proved. However, these mutants were more sensitive to UV radiation and alkyliernden substances, so it is believed that the DNA polymerase I repair mainly takes tasks. Polymerase III, which performs the actual replication in E. coli is composed of seven subunits per bacterial cell and is in very few copies before.

In mammals, five DNA polymerases occur. DNA polymerase α, β, γ, δ and ε. The key for replication are here seemingly polymerases δ and ε, which are characterized by high processivity and proofreading distinguished ( proofreading ). The polymerases α and β, however, show only low processivity and no proofreading function. The polymerase γ occurs only in mitochondria.

Biological Significance

DNA polymerases are essential for DNA replication. They allow the faithful copying of the genetic information in the form of DNA, thus a critical step in the propagation and reproduction of living organisms. DNA polymerases also play in processes associated with the repair of DNA, an important role.

Biotechnological importance

DNA polymerases in the laboratory are often used for the polymerase chain reaction and related methods (e.g., RT-PCR, qPCR), wherein the nick translation, random priming and in DNA sequencing. Thereby a plurality of different, sometimes modified by protein engineering thermostable DNA polymerases is used (eg, Taq polymerase). In addition to bringing a high temperature stability of thermostable DNA polymerases Archaean origin with a proofreading function ( proof-reading ), since there is to be no change in the DNA generated during the PCR.