Investigation of the clamp loading mechanism from biochemical and 3D structural perspectives

Abstract

In highly processive genomic DNA duplication, ring-shaped sliding clamps and clamp loader ATPases are essential factors. Proliferating cell nuclear antigen (PCNA) and replication factor C (RFC) play crucial roles as the clamp loader and the clamp, respectively. Pyrococcus furiosus RFC and PCNA were used to reconstruct a clamp loading complex with a synthetic template-primer DNA, and the electron microscopic structure of a P. furiosus clamp loading complex at 12 Å resolution was obtained. This complex exhibits excellent fitting of each atomic structure of RFC, PCNA, and the primed DNA. The complex appears to represent the intermediate, in which the PCNA ring is kept open with a distorted spring washer-like conformation. A model of the clamp loading mechanism is proposed, based on the 3D structure of the complex, along with information from biochemical analyses.