Overproduction and purification of RFC-related clamp loaders and PCNA-related clamps from Saccharomyces cerevisiae

Abstract

The replication clamp PCNA and its loader RFC (Replication Factor C) are central factors required for processive replication and coordinated DNA repair. Recently, several additional related clamp loaders have been identified. These alternative clamp loaders contain the small Rfc2-5 subunits of RFC, but replace the large Rfc1 subunit by a pathway-specific alternative large subunit, Rad24 for the DNA damage checkpoint, Ctf18 for the establishment of sister chromatid cohesion, and Elg1 for a general function in chromosome stability. In order to define biochemical functions for these loaders, the loaders were overproduced in yeast and purified at a milligram scale. To aid in purification, the large subunit of each clamp loader was fused to a GST-tag that, after purification could be easily removed by a rhinoviral protease. This methodology yielded all clamp loaders in high yield and with high enzymatic activity. The yeast 9-1-1 checkpoint clamp, consisting of Rad17, Mec3, and Ddc1, was overproduced and purified in a similar manner.