Exploiting the biotechnological potential of the unusual archaeal PolD DNA polymerase by unravelling its specificities at the structural level – ARCHAPOL

DNA polymerases (DNAPs) are molecular motors directing the synthesis of DNA from nucleotides. On the basis of their amino acid sequence and structural analysis, DNAPs have been classified into seven families, A, B, C, D, X, Y and reverse transcriptases. In addition to their fundamental biological functions, DNAPs are versatile tools used in important molecular biology core technologies. The best known DNAP-based biotechnology application is the polymerization chain reaction (PCR). In the PCR, DNA amplification is performed by thermostable DNAPs, which have different characteristics, such as specificity, processivity, fidelity, resistance to contaminants ... As nucleic acid analysis by PCR moves toward clinical diagnostics and forensics, there is a constant need for DNAPs capable of amplifying DNA from clinical samples such as tissue, blood, body fluids.

Thermostable DNAPs marketed for PCR invariably are either family-A DNAPs from thermophilic and hyperthermophilic Bacteria, family-B and family-Y DNAPs from the hyperthermophilic Archaea. Recently, a novel family (D-family) of archaeal thermostable DNAP, named PolD, was discovered and shown to have significant commercial value in PCR technology. In particular, PolD from Pyrococcus abyssi showed not only greater resistance to high denaturation temperatures than the popular Taq during cycling, but also superior tolerance to the presence of potential inhibitors (including ions and detergents), and is completely resistant to haemoglobin. In addition, PolD shows among the highest tolerance to calcium ions compared to other thermostable DNAPs, thereby placing PolD as a suitable enzyme in the amplification of food (e.g., milk and cheese) and human samples (e.g., teeth and bones). PolD is composed of a large catalytic subunit (DP2) and a smaller subunit with proofreading exonuclease activity (DP1). We have determined the first crystal structures of both individual DP1 and DP2 catalytic subunits of PolD from P. abyssi, and showed that it is structurally unrelated to any other DNAP family. However, the DP1 and DP2 crystal structures were obtained separately and do not provide a comprehensive description of the molecular mechanisms of DNA polymerization and proofreading by PolD. Indeed, the DNAP active site is located at the interface between the DP1 and DP2 subunits, and the interaction between both subunits is essential for the full activity of PolD. Unlike other DNAPs used in PCR, most of whose crystal structures have been solved in complex with various DNA substrates, the molecular basis of DNA and nucleotide-recognition by PolD remains poorly characterized. Elucidating the specificities of the PolD active site at the molecular level is required in order to fully exploit the biotechnological potential of this unusual thermostable DNAP.

Using a multi-disciplinary approach combining electron microscopy, X-ray crystallography, and Small-angle X-ray/Neutron Scattering, we aim to determine the DNA-bound structures of the native full DP1-DP2 PolD complex in both elongation and proof-reading modes, as well as in complex with the proliferating cell nuclear antigen (PCNA), a key actor of the replication fork that has been shown to considerably improve the processivity of PolD. Taking advantage of this structural information, we will engineer PolD variants showing an optimized coupling to PCNA in order to improve both its processivity and DNA sensitivity under PCR conditions, as well as, using directed-evolution, heparin-tolerant PolD variants. These PolD variants will be ideally suited for cutting-edge PCR applications in forensics and clinical diagnostics. This study will also clarify whether PolD that constitutes a structurally-distinct class of DNAPs has evolved specific mechanisms of polymerization, DNA-binding, nucleotide selection, or did follow a converging evolution with the other classes of DNA polymerases.