PDBsum entry 3e0d

Transferase/DNA

PDB id: 3e0d

Contents

Protein chains

1148 a.a.

DNA/RNA

Ligands

DTP ×2

Metals

_CA ×2

References listed in PDB file

Key reference

Title

Insights into the replisome from the structure of a ternary complex of the DNA polymerase III alpha-Subunit.

Authors

R.A.Wing, S.Bailey, T.A.Steitz.

Ref.

J Mol Biol, 2008, 382, 859-869. [DOI no: 10.1016/j.jmb.2008.07.058]

PubMed id

18691598

Abstract

The crystal structure of the catalytic alpha-subunit of the DNA polymerase III (Pol IIIalpha) holoenzyme bound to primer-template DNA and an incoming deoxy-nucleoside 5'-triphosphate has been determined at 4.6-A resolution. The polymerase interacts with the sugar-phosphate backbone of the DNA across its minor groove, which is made possible by significant movements of the thumb, finger, and beta-binding domains relative to their orientations in the unliganded polymerase structure. Additionally, the DNA and incoming nucleotide are bound to the active site of Pol IIIalpha nearly identically as they are in their complex with DNA polymerase beta, thereby proving that the eubacterial replicating polymerase, but not the eukaryotic replicating polymerase, is homologous to DNA polymerase beta. Finally, superimposing a recent structure of the clamp bound to DNA on this Pol IIIalpha complex with DNA places a loop of the beta-binding domain into the appropriate clamp cleft and supports a mechanism of polymerase switching.

Figure 2.
Fig. 2. Crystal structure of a replicating complex of T. aquaticus PolIIIα. (a) A view of the crystal structure of the replicating complex of T. aquaticus PolIIIα. Domains are labeled as follows: PHP nuclease, yellow; palm, pink; thumb, light green; index finger, light blue; middle finger, dark blue; β-binding, orange; and CTD, red. The template and primer strands are represented as spheres and shown in wheat and white, respectively. The primer-terminal 3′ base is depicted with blue spheres, and the incoming nucleotide is depicted with red spheres. PolIIIα is depicted in a cartoon representation. The density for the downstream DNA template strand is represented in mesh. (b) Close-up of the active site of PolIIIα. PolIIIα is depicted using a surface representation. The DNA substrate is depicted as sticks and shown as in the left panel. The incoming nucleotide and primer-terminal 3′ base are shown in red and blue, respectively. Key active-site residues are labeled as follows: D463, D465, and D618 are shown in red; K616 is in yellow; S426 is in green; and R767 is in blue. The active-site helix is shown in magenta.

Figure 3.
Fig. 3. Interactions with the DNA substrate. (a) Surface representation of interactions between the thumb domain of PolIIIα and the DNA substrate in a cartoon representation. Green surfaces indicate the thumb domain, with blue surfaces representing residues proposed to interact with the DNA substrate. Yellow surfaces represent the PHP nuclease domain. The polymerase active site is seen on the left as demarcated by the blue stick representation of the primer-terminal 3′ base (ddC) and the red stick representation of the incoming nucleotide (dATP). (b) Surface representation of interactions between the β-binding domain of PolIIIα and the DNA substrate in a cartoon representation. Orange surfaces indicate the β-binding domain with three key loops implicated in DNA binding labeled as follows: Taq927–Taq923, blue; HhH motif Taq892–Taq910, cyan; and Taq846–Taq852, purple. Positively charged residues that are within proximity to interact with the sugar–phosphate backbone are labeled, and their positions are indicated with white arrows. An additional arrow points to the location of the polymerase active site. (c) The observed DNA substrate of PolIIIα is depicted using a cartoon representation with the primer strand shown in white and the template strand shown in wheat. Idealized B-form DNA aligned with the DNA substrate prior to bending is displayed in a surface representation. Black lines indicate the helical axes of the DNA substrate (outside of the active site) and the idealized B-form DNA. The primer-terminal 3′ base is represented in blue sticks. The loops of the thumb domain (Taq492–Taq499 and Taq569–Taq575) that induce DNA bending are shown in green and represented as a surface.

The above figures are reprinted by permission from Elsevier: J Mol Biol (2008, 382, 859-869) copyright 2008.