Pfu polymerase

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DNA polymerase ( Pyrococcus furiosus )
DNA polymerase (Pyrococcus furiosus)

Existing structural data : 2jgu , 3a2f

Mass / length primary structure 775 amino acids
Identifier
External IDs
Enzyme classification
EC, category 2.7.7.7 ,  nucleotidyl transferase
Response type Replication
Substrate Deoxyribonucleoside triphosphate + DNA n
Products Diphosphate + DNA n + 1

The Pfu polymerase is an enzyme from the thermophilic archaea Pyrococcus furiosus . It is a thermostable DNA polymerase with a proof-reading function that is used in biochemical laboratory work in the polymerase chain reaction (PCR).

As with all DNA polymerases, DNA synthesis takes place in the 5 '→ 3' direction. The proofreading function of the Pfu polymerase (3 '→ 5' exonuclease activity ) ensures that the newly synthesized DNA strand is continuously checked for mismatches, which are corrected immediately with the removal of the faulty nucleotide and the incorporation of the correct base-pairing nucleotide. The Pfu is mainly used in molecular biology for those PCR reactions that have to deliver DNA amplificates with exact sequence. For the other PCR operations, the much cheaper and faster, but less accurate Taq polymerase is sufficient. In contrast to Taq polymerases, the Pfu does not generate 3 'overhangs from adenosyl residues , but rather provides " blunt ends ". This enables the cloning of blunt-end cut DNA.

The read-ahead function of Pfu polymerase is characterized by the advance recognition of uracil in DNA strands. Uracil can be formed by deamination of cytosine and would result in base pairing with adenine (A: U instead of G: C) - it is not normally found in DNA. The specific function of Pfu polymerase is to stop DNA synthesis when uracil is recognized. Other repair enzymes then correct the DNA. When using Pfu polymerase in a polymerase chain reaction (PCR), uracil or dUTP leads to a reduced synthesis rate, which is referred to as dUTP poisoning ( dUTP poisoning ). In contrast, dUTPases are added. Alternative thermostable DNA polymerases that are used in PCR are, for example, Pwo polymerase (with proof-reading ) and Taq polymerase (without proof-reading ).

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