DNA Polymerase I

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Structures of Klentaq1 in its closed and open forms

Pol I replicates DNA with high fidelity. How does it do so? Gabriel Waksman answered this question by crystallizing the C-terminal domain of Taq polymerase (Klentaq1) with an 11-bp DNA that had a GGAAA-5' overhang at the 5' end of its template strand[3]. The crystals were then soaked in solution containing 2',3'-dideoxy-CTP (ddCTP), which lacks a 3'-OH group, and hence terminates replication after its incorporation at the 3' end of the primer strand. The X-ray structure of these crystals revealed that a ddC residue had been covalently linked to the 3' end of the primer strand, where it formed a Watson–Crick base pair with the 3' G on the template overhang, thus demonstrating the Klentaq1 is enzymatically active in the crystal. In addition, a ddCTP molecule occupied the enzyme's active site, where it formed a Watson–Crick base pair with the template's next G.

Klentaq1–Closed conformation (3ktq)

Klentaq1–Open conformation (2ktq)

Here, Klentaq1's N-terminal, palm, fingers and thumb domains are yellow, magenta, green, and blue, respectively. The DNA is drawn in stick form colored according to atom type (template C cyan, primer C green, N blue, O red, and P orange).

In the structure on the left, the crystal had been soaked in a solution of dideoxy-CTP (ddCTP), which the enzyme had added to the 3' end of the primer chain (shown in space-filling form with C green), where it forms a base pair with the a template G. This terminates further primer extension due to the absence of a 3'-OH group at the 3' end of the primer strand. Nevertheless, a ddCTP (shown in space-filling form with C yellow) binds to the enzyme active site at the 3' end of the primer in a base pair with a template G as if it were preparing to add to the 3' end of the primer. In the structure on the right, the ddCTP in the enzyme's active site had been depleted by soaking the crystal in a ddCTP-frree solution. Comparison of these two structures reveals that the structure on the left, the so-called closed conformation, differs from the that on the right, the so-called open conformation, by a hinge-like motion of the fingers domain away from the polymerase active site. The rest of the protein remains very nearly unchanged. This is more readily seen in the (left, in which, for technical reasons, the ddCTP in the closed conformation is not shown).

This, together with other experimental measurements, indicates that Klentaq1 rapidly samples the available dNTPs in its open conformation, but only when it binds the correct dNTP in a Watson–Crick pairing with the template base does it form the catalytically competent closed conformation. In addition, note how the template G that base pairs with the ddCTP in the closed conformation, moves away from the active site in the open conformation, in which it has no base pairing partner.

A closeup of the active site region in the (right) reveals that the side chain of the conserved Tyr 671 (colored with C pink) is stacked on top of the template G that forms a base pair with the bound ddCTP, where it apparently participates in verifying that a Watson–Crick base pair has formed. In the (left), Tyr 671, which is part of the fingers domain, has moved aside, presumably to permit the active site to form about the incoming dNTP (satisfy yourself that the Tyr 671 side chain is stacked on the template G in the open form but not in the closed form).

3D structures of DNA polymerase

DNA polymerase

Additional Resources

For additional information, see: DNA Replication, Repair, and Recombination
For additional information, see: Nucleic Acids


  1. Beese LS, Derbyshire V, Steitz TA. Structure of DNA polymerase I Klenow fragment bound to duplex DNA. Science. 1993 Apr 16;260(5106):352-5. PMID:8469987
  2. Friedman AM, Fischmann TO, Steitz TA. Crystal structure of lac repressor core tetramer and its implications for DNA looping. Science. 1995 Jun 23;268(5218):1721-7. PMID:7792597
  3. Li Y, Korolev S, Waksman G. Crystal structures of open and closed forms of binary and ternary complexes of the large fragment of Thermus aquaticus DNA polymerase I: structural basis for nucleotide incorporation. EMBO J. 1998 Dec 15;17(24):7514-25. PMID:9857206 doi:http://dx.doi.org/10.1093/emboj/17.24.7514

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