Sandbox dvoet/DNA polymerase
From Proteopedia
DNA polymerase I
DNA replication is catalyzed by DNA polymerase. All cells express several different DNA polymerases that variously participate in the several aspects of DNA replication and in the repair of damaged DNA. DNA polymerases catalyze the reaction (DNA)n residues + dNTP → (DNA)n+1 residues + PPi, where dNTP is the deoxynucleoside triphosphate whose base is complementary to a base on the strand being copied, the so-called template strand. In addition, DNA polymerases cannot initiate replication by linking together two dNTPs, but rather, can only link the incoming nucleotide to a terminal 3'-OH group on an existing polynucleotide strand, the so-called primer strand, thereby forming a 3' → 5' phosphodiester bond between successive deoxynucleotides.
If DNA polymerase can only add nucleotides to a pre-existing primer strand, how can the primer be synthesized? The answer is that the initial primer is a short RNA strand that is complementary to the a portion of the template strand and which is synthesized by an RNA polymerase known as primase. This enzyme catalyzes a reaction similar to that catalyzed by DNA polymerase but uses NTPs rather than dNTPs. However primase, as can all RNA polymerases, does not require a primer to initiate polynucleotide synthesis; it can do so by linking together two NTPs in a 3' → 5' linkage.
The first known DNA polymerase, an E. coli enzyme now known as DNA polymerase I or Pol I, was discovered and characterized in 1957 by Arthur Kornberg (who received the Nobel prize for this work). Pol I has three active sites:
- 1. A DNA polymerase.
- 2. A 3' → 5' exonuclease, that hydrolyzes off mispaired nucleotides at the 3' end of the growing polynucleotide [(DNA)n residues + H2O → (DNA)n-1 residues + dNMP] and hence provides Pol I with the ability to proofread and edit its mistakes.
- 3. A 5' → 3' exonuclease, whose central role is to remove the RNA primers (although it also participates in DNA repair processes), which the polymerase function then replaces with DNA.
These active sites occupy different regions of Pol I. In fact, mild treatment of Pol I by proteases such as trypsin, cleaves Pol I into two catalytically active fragments. The N-terminal fragment contains the 5' → 3' exonuclease function, whereas the larger, C-terminal fragment, which is known as Klenow fragment, contains both the polymerase and the 3' → 5' exonuclease functions.
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