TY - JOUR
T1 - Overproduction and Purification of RFC-Related Clamp Loaders and PCNA-Related Clamps from Saccharomyces cerevisiae
AU - Bylund, Göran O.
AU - Majka, Jerzy
AU - Burgers, Peter M.J.
N1 - Funding Information:
This work was supported in part by Grant GM32431 from the National Institutes of Health. G.O.B. was supported in part by a stipend from the Washington University‐Umeå University Exchange program.
PY - 2006
Y1 - 2006
N2 - 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.
AB - 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.
UR - http://www.scopus.com/inward/record.url?scp=33745203951&partnerID=8YFLogxK
U2 - 10.1016/S0076-6879(05)09001-4
DO - 10.1016/S0076-6879(05)09001-4
M3 - Review article
C2 - 16793392
AN - SCOPUS:33745203951
SN - 0076-6879
VL - 409
SP - 1
EP - 11
JO - Methods in Enzymology
JF - Methods in Enzymology
ER -