Three-dimensional electron microscopy of the clamp loader small subunit from Pyrococcus furiosus

An archaeal clamp loader, replication factor C (RFC), consists of two prote ins, the small subunit (RFCS) and large subunit (RFCL), whose sequences are both highly homologous to those of the eukaryotic RFC components. We have investigated the oligomeric structure of RFCS from Pyrococcus furiosus by e lectron microscopy using single-particle analysis. RFCS forms mostly ring-s haped hexamers at pH 9.0, although it tends to form C-shaped tetramers or p entamers at a lower pH (pH 5.5). The three-dimensional (3D) structure of th e RFCS hexamer was obtained by random conical tilt reconstruction at 24.0-A ngstrom resolution. RFCS forms a hexameric ring with outer and inner diamet ers of 117 and 27 Angstrom, respectively, and with a height of about 55 Ang strom. The six subunits are arranged in a twisted manner with a sixfold sym metry around the channel. The 3D map revealed that the six subunits are arr anged in a head-to-tail configuration. Although the RFC complex consists of RFCS and RFCL in vivo, RFCS alone, together with PCNA, substantially enhan ced the DNA synthesizing activity of P. furiosus DNA polymerase I in vitro. The 3D reconstruction of RFCS with catalytic activity provides important i nsights into the organization mechanism and the functional state of the RFC complex. (C) 2001 Academic Press.