Single Molecule Measurements Reveal Conformational Transitions During DNA Clamp Loading and Unloading

Abstract

Proliferation cell nuclear antigen (PCNA) is a DNA clamp, playing an important role of providing a ‘‘platform’’ for various enzymes during DNA replication. The loading of the closed trimeric ring of PCNA into duplex DNA requires the ATP-dependent activity of replication factor C (RFC) complex. The unloading of PCNA from chromatin is crucial for the regulation of replication process and maintaining genomic stability and it was recently found that ATAD5 protein is complexed with RFC-like complex (RLC) to get involved in the unloading of PCNA. However, the molecular mechanisms of PCNA loading and unloading processes have remained poorly understood. Here, we report direct observation of the loading and unloading dynamics of human PCNA driven by RFC and ATAD5-RLC complexes, respectively, by single molecule fluorescence resonance energy transfer measurements. Distinct conformational stages during PCNA loading were clearly detected that represent open and closed conformations of PCNA trimer and another loading intermediate that possibly triggers the dissociation of RFC complex. The unloading of PCNA occurred upon binding ATAD5-RLC complex through an intermediate conformation, but not requiring the hydrolysis of ATP, which is later employed to recycle PCNA and ATAD5-RLC complexes. Our findings present a mechanistic model of the clamp loading/unloading dynamics and bring implications on the mechanism controlling the exclusive functions of RFC and ATAD5-RLC complexes in the loading and unloading processes, respectively. It further provides a platform to study how PCNA controls the exchange of various replication enzymes.