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ATP utilization by yeast replication factor C. III. The ATP-binding domains of Rfc2, Rfc3, and Rfc4 are essential for
S L Schmidt1, X V Gomes, P M Burgers
1Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine, St. Louis, Missouri 63110, USA.
The Journal of Biological Chemistry
|July 4, 2001
Summary
Mutations in yeast replication factor C (RFC) subunits RFC2 and RFC3 severely impaired ATPase and DNA binding activities. RFC4 mutations only affected clamp loading at high ATP concentrations, while RFC1 mutations increased proteolysis susceptibility.
Area of Science:
- Molecular Biology
- Biochemistry
- Yeast Genetics
Background:
- Replication Factor C (RFC) is a crucial clamp loader complex essential for DNA replication.
- The conserved lysine in the Walker A motif of the ATP-binding domain is vital for RFC function.
- Understanding the roles of individual RFC subunits requires targeted mutagenesis and biochemical analysis.
Purpose of the Study:
- To investigate the functional significance of the conserved lysine in the Walker A motif of yeast RFC subunits (RFC1, RFC2, RFC3, RFC4).
- To elucidate the impact of specific mutations on RFC complex stability, DNA binding, ATPase activity, and clamp loading function.
Main Methods:
- Site-directed mutagenesis was used to introduce lysine-to-glutamic acid (K-E) and lysine-to-arginine (K-R) mutations in yeast RFC subunits.
- Recombinant RFC complexes with truncated Rfc1 and single mutant subunits were overexpressed in E. coli.
- Biochemical assays were performed to assess PCNA interaction, DNA binding, ATPase activity, and clamp loading activity.
Main Results:
- RFC complexes with rfc1-K359E mutation showed wild-type replication and ATPase activity but increased proteolysis susceptibility.
- rfc2-K71E and rfc3-K59E mutations severely impaired ATPase, DNA binding, and clamp loading activities.
- rfc4-K55E mutation affected clamp loading only at very high ATP concentrations.
- Conservative rfc2-K71R and rfc3-K59R mutations resulted in milder clamp loading defects, partially or fully suppressed by high ATP.
Conclusions:
- The conserved lysine in the Walker A motif plays distinct roles in different RFC subunits.
- RFC2 and RFC3 lysine residues are critical for ATPase, DNA binding, and clamp loading.
- RFC4 lysine is important for efficient clamp loading, particularly under low ATP conditions.
- RFC1 lysine's role appears less critical for core enzymatic activities but influences complex stability.