E. coli and S. cerevisiae clamp loaders, γ complex and RFC, actively close clamps

Abstract

Sliding clamps and their respective clamp loaders perform integral functions in DNA metabolism and are therefore found throughout all kingdoms of life. Clamp loaders, members of the AAA+ ATPase family, use ATP binding and hydrolysis to load sliding clamps onto DNA where the clamps then serve as a platform for enzymes that act on DNA. In E. coli, the β clamp is loaded by the γ complex clamp loader, and in S. cerevisiae, PCNA is loaded by the RFC clamp loader. Using fluorescent assays developed in our laboratory to report on clamp closing and release, we have shown that the rate of clamp closing is about 10 times faster than the rate of clamp release for both systems, indicating that these clamp loaders actively close clamps. When ATP hydrolysis is blocked, the closing and release rates become similar, indicating that the mechanism of closing before release is ATP hydrolysis‐dependent. ATP hydrolysis experiments were performed using fluorescently labeled phosphate binding protein to determine the rate of ATP hydrolysis and a temporal correlation between ATP hydrolysis and clamp closing. These experiments identify a common mechanistic feature in the clamp loading reaction and help to understand how these enzymes efficiently load clamps for replication and repair.This work supported by NIH grant GM 082849 and 5 T32 CA009126‐32 Training Grant in Cancer Biology