Abstract
BackgroundDNA polymerase D (PolD) is the representative member of the D family of DNA polymerases. It is an archaea-specific DNA polymerase required for replication and unrelated to other known DNA polymerases. PolD consists of a heterodimer of two subunits, DP1 and DP2, which contain catalytic sites for 3′-5′ editing exonuclease and DNA polymerase activities, respectively, with both proteins being mutually required for the full activities of each enzyme. However, the processivity of the replicase holoenzyme has additionally been shown to be enhanced by the clamp molecule proliferating cell nuclear antigen (PCNA), making it crucial to elucidate the interaction between PolD and PCNA on a structural level for a full understanding of its functional relevance. We present here the 3D structure of a PolD-PCNA-DNA complex from Thermococcus kodakarensis using single-particle cryo-electron microscopy (EM).ResultsTwo distinct forms of the PolD-PCNA-DNA complex were identified by 3D classification analysis. Fitting the reported crystal structures of truncated forms of DP1 and DP2 from Pyrococcus abyssi onto our EM map showed the 3D atomic structural model of PolD-PCNA-DNA. In addition to the canonical interaction between PCNA and PolD via PIP (PCNA-interacting protein)-box motif, we found a new contact point consisting of a glutamate residue at position 171 in a β-hairpin of PCNA, which mediates interactions with DP1 and DP2. The DNA synthesis activity of a mutant PolD with disruption of the E171-mediated PCNA interaction was not stimulated by PCNA in vitro.ConclusionsBased on our analyses, we propose that glutamate residues at position 171 in each subunit of the PCNA homotrimer ring can function as hooks to lock PolD conformation on PCNA for conversion of its activity. This hook function of the clamp molecule may be conserved in the three domains of life.
Highlights
Deoxyribonucleic acid (DNA) polymerase D (PolD) is the representative member of the D family of DNA polymerases
Reconstitution of the PolD-proliferating cell nuclear antigen (PCNA)-DNA complex We reconstituted and purified the PolD-PCNA-DNA complex using DNAs listed in Additional file: Figure S1 by gel filtration chromatography (Additional file: Figure S2A-D)
Here, we present the 3D structure of PolD, the archaealspecific DNA polymerase, complexed with a primed DNA and PCNA
Summary
DNA polymerase D (PolD) is the representative member of the D family of DNA polymerases It is an archaea-specific DNA polymerase required for replication and unrelated to other known DNA polymerases. PolD consists of a heterodimer of two subunits, DP1 and DP2, which contain catalytic sites for 3′-5′ editing exonuclease and DNA polymerase activities, respectively, with both proteins being mutually required for the full activities of each enzyme. Among the archaeal DNAPs, DNA polymerase Bs (PolBs), especially those found in hyperthermophilic archaea, such as Pyrococcus furiosus PolB (PfuPolB) [5] or Thermococcus kodakarensis PolB (TkoPolB) [6], have been structurally and biochemically studied in detail and are widely applied in biotechnology for PCR because of their extreme thermostability and outstanding fidelity [7]. The PolD holoenzyme consists of two subunits, DP1 and DP2, which contain the catalytic sites of the 3′–5′ exonuclease and the 5′–3′ DNA polymerase, respectively
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