Overproduction and affinity purification of Saccharomyces cerevisiae replication factor C.
Yeast replication factor C (RF-C) is a heteropentamer encoded by the RFC1-5 genes. RF-C activity in yeast extracts was overproduced about 80-fold after induction of a strain containing all five genes on a single plasmid, with expression of each gene placed under control of the galactose-inducible GAL1-10 promoter. This strongly indicates that overexpression of the five known RFC genes is sufficient for overproduction of RF-C. Overexpression of all five genes was also necessary to achieve overproduction of RF-C as omission of any single gene from the plasmid gave uninduced, i.e. normal cellular levels of RF-C. The interaction between RF-C and proliferating cell nuclear antigen (PCNA) was studied with PCNA-agarose beads. Binding of RF-C to PCNA-agarose beads is negligible in buffers containing 0.3 M NaCl. However, addition of Mg-ATP to the binding buffer caused strong binding of RF-C to the beads even at 0.8 M NaCl. Binding of ATP, but not its hydrolysis, was required for the strong binding mode as nonhydrolyzable analogs were also effective. The existence of two distinct binding modes between PCNA and RF-C was used as the key step in a greatly improved procedure for the purification of RF-C. RF-C from the overproduction strain purified by this procedure was essentially homogeneous and had a severalfold higher specific activity than RF-C preparations that had previously been purified through multicolumn procedures.
Gerik
K J
KJ
Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine, St. Louis, Missouri 63110, USA.
Gary
S L
SL
Burgers
P M
PM
D000255:Adenosine Triphosphate / Q000378:metabolism; D002846:Chromatography, Affinity; D004261:DNA Replication; D004268:DNA-Binding Proteins / Q000302:isolation & purification*; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli; D005690:Galactose / Q000378:metabolism; D018398:Homeodomain Proteins; D015778:Minor Histocompatibility Antigens; D018809:Proliferating Cell Nuclear Antigen; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000302:isolation & purification*; D011887:Raffinose / Q000378:metabolism; D051818:Replication Protein C; D012097:Repressor Proteins; D012441:Saccharomyces cerevisiae; D029701:Saccharomyces cerevisiae Proteins
C096816:BCL2-related protein A1; D004268:DNA-Binding Proteins; D018398:Homeodomain Proteins; C096063:MATA1 protein, S cerevisiae; D015778:Minor Histocompatibility Antigens; D018809:Proliferating Cell Nuclear Antigen; D019253:Proto-Oncogene Proteins c-bcl-2; D012097:Repressor Proteins; D029701:Saccharomyces cerevisiae Proteins; D000255:Adenosine Triphosphate; D051818:Replication Protein C; D011887:Raffinose; D005690:Galactose