ATP utilization by yeast replication factor C. III. The ATP-binding domains of Rfc2, Rfc3, and Rfc4 are essential for DNA recognition and clamp loading.
The conserved lysine in the Walker A motif of the ATP-binding domain encoded by the yeast RFC1, RFC2, RFC3, and RFC4 genes was mutated to glutamic acid. Complexes of replication factor C with a N-terminal truncation (Delta2-273) of the Rfc1 subunit (RFC) containing a single mutant subunit were overproduced in Escherichia coli for biochemical analysis. All of the mutant RFC complexes were capable of interacting with PCNA. Complexes containing a rfc1-K359E mutation were similar to wild type in replication activity and ATPase activity; however, the mutant complex showed increased susceptibility to proteolysis. In contrast, complexes containing either a rfc2-K71E mutation or a rfc3-K59E mutation were severely impaired in ATPase and clamp loading activity. In addition to their defects in ATP hydrolysis, these complexes were defective for DNA binding. A mutant complex containing the rfc4-K55E mutation performed as well as a wild type complex in clamp loading, but only at very high ATP concentrations. Mutant RFC complexes containing rfc2-K71R or rfc3-K59R, carrying a conservative lysine --> arginine mutation, had much milder clamp loading defects that could be partially (rfc2-K71R) or completely (rfc3-K59R) suppressed at high ATP concentrations.
Schmidt
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SL
Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine, St. Louis, Missouri 63110, USA.
Gomes
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XV
Burgers
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PM
D000255:Adenosine Triphosphate / Q000378:metabolism*; D000595:Amino Acid Sequence; D001665:Binding Sites; D004247:DNA / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000378:metabolism*; D005656:Fungal Proteins / Q000378:metabolism*; D006801:Humans; D008969:Molecular Sequence Data; D018809:Proliferating Cell Nuclear Antigen / Q000378:metabolism; D021122:Protein Subunits; D051818:Replication Protein C; D029701:Saccharomyces cerevisiae Proteins; D013329:Structure-Activity Relationship; D015003:Yeasts / Q000737:chemistry*
D004268:DNA-Binding Proteins; D005656:Fungal Proteins; D018809:Proliferating Cell Nuclear Antigen; D021122:Protein Subunits; C087491:RFC2 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D000255:Adenosine Triphosphate; D004247:DNA; D051818:Replication Protein C