DOI 10.1371/journal.pbio.3000122 PolD is an archaeal replicative DNA polymerase (DNAP) made of a proofreading exonuclease subunit (DP1) and a larger polymerase catalytic subunit (DP2). Recently, we reported the individual crystal structures of the DP1 and DP2 catalytic cores, thereby revealing that PolD is an atypical DNAP that has all functional properties of a replicative DNAP but with the catalytic core of an RNA polymerase (RNAP). We now report the DNA-bound cryo–electron microscopy (cryo-EM) structure of the heterodimeric DP1–DP2 PolD complex from Pyrococcus abyssi, revealing a unique DNA-binding site. Comparison of PolD and RNAPs extends their structural similarities and brings to light the minimal catalytic core shared by all cellular transcriptases. Finally, elucidating the structure of the PolD DP1–DP2 interface, which is conserved in all eukaryotic replicative DNAPs, clarifies their evolutionary relationships with PolD and sheds light on the domain acquisition and exchange mechanism that occurred during the evolution of the eukaryotic replisome.
| File | Pages | Size | Access | |
|---|---|---|---|---|
Publisher's official version | 28 | 7 Mo | ||
S1 Table. Values of cryo-EM data collection and 3D reconstruction. | 1 | 175 Ko | ||
S2 Table. Data collection and refinement statistics. | 1 | 76 Ko | ||
S1 Text. Details on anisotropy and Kd calculations for the DNA-binding assays by steady-state fluorescence anisotropy. | 1 | 178 Ko | ||
S2 Text. Details on the structure determination of the DP1 H451A proofreading-deficient variant by X-ray crystallography. | 2 | 84 Ko | ||
S1 Fig. FSC curves and local resolutions. | 1 | 176 Ko | ||
S2 Fig. Crystal structure of the proofreading-deficient H451A DP1 variant. | 1 | 595 Ko | ||
S3 Fig. Purification of the DP1ΔN(144–619)–DP2CTD(1096–1195) complex. | 1 | 115 Ko | ||
S4 Fig. Structure-based alignment of the shared structural elements within the catalytic cores of PolD, DNA-dependent RNA polymerases, and RNA-dependent RNA polymerases. | 1 | 241 Ko | ||
S5 Fig. Image processing of the cryo-EM 3D reconstruction of PolD. | 1 | 632 Ko | ||
S6 Fig. List of rigid-body groups used for real-space refinement. | 1 | 419 Ko | ||
S1 Data. | - | 208 Ko |
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