Expression, purification and preliminary X-ray analysis of proliferating cell nuclear antigen from the archaeon Thermococcus thioreducens.

Proliferating cell nuclear antigen (PCNA) is a DNA sliding clamp which confers processivity on replicative DNA polymerases. PCNA also acts as a sliding platform that enables the association of many DNA-processing proteins with DNA in a non-sequence-specific manner. In this investigation, the PCNA from the hyperthermophilic archaeon Thermococcus thioreducens (TtPCNA) was cloned, overexpressed in Escherichia coli and purified to greater than 90% homogeneity. TtPCNA crystals were obtained by sitting-drop vapor-diffusion methods and the best ordered crystal diffracted to 1.86 A resolution using synchrotron radiation. The crystals belonged to the hexagonal space group P6(3), with unit-cell parameters a = b = 89.0, c = 62.8 A. Crystals of TtPCNA proved to be amenable to complete X-ray analysis and future structure determination.