Sandbox dvoet/DNA polymerase

DNA polymerase IDNA 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} + PP_i, 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 and subtilisin, cleaves Pol I into two catalytically active fragments. The N-terminal fragment (residues 1-323) contains the 5' → 3' exonuclease function, whereas the larger, C-terminal fragment (residues 324-928), which is known as the Klenow fragment, contains both the polymerase and the 3' → 5' exonuclease functions.

spinqualitylabelsall models Klenow–DNA Show:Asymmetric Unit Biological Assembly Drag the structure with the mouse to rotate   Export Animated Image

Thomas Steitz determined the X-ray structure of Klenow fragment in complex with a 13-nucleotide (nt) primer strand and a 10-nt template strand (the primer strand is the strand that is synthesized by the polymerase as the complement of the template strand; the entire DNA is often referred to as primer−template DNA). Here Klenow fragment is shown in ribbon form colored in rainbow order from its N-terminus (blue) to its C-terminus (red). The DNA is drawn in stick form and colored according to atom type with template C cyan, primer C magenta, N blue, O red, and P orange and with an orange rod connecting successive P atoms in each strand. The 3' → 5' exonuclease active site at the N-terminal end of the protein is marked by a Zn²⁺ ion (gray sphere). 'The arrangement of the polymerase's three domains is reminiscent of a right hand grasping a rod (the DNA) and hence, from N- to C-terminus, they are named “palm“, "fingers", and "thumb". The polymerase's active site is located in the palm domain near the cleft between the fingers and thumb domains. All DNA polymerases of known structure have a similar spatial arrangements of fingers, thumb, and palm domains, even though, in many cases, they have no recognizable sequence similarity with Pol I and the structure of their fingers, thumb, and palm domains bear no resemblance to those of Pol I.

spinqualitylabelsall models Klenow–DNA closeup Show:Asymmetric Unit Biological Assembly Drag the structure with the mouse to rotate   Export Animated Image

The X-ray structure is that of an editing complex, that is, the 3' end of the primer strand, the end that is elongated by the polymerase, occupies the 3'→5' exonuclease active site. This is more clearly seen in a in which the the rods connecting successive P atoms have been removed for clarity. Note that the base pair closest to the polymerase active site, a G·C, has opened up to enable the 3' end of the primer strand to reach the exonuclease active site. Click here to .