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ATP hydrolysis catalyzed by human replication factor C requires participation of multiple subunits
1Program in Molecular Biology, William Randolph Hearst Laboratory of Radiation Biology, Memorial Sloan-Kettering Cancer Center, 1275 York Avenue/ Box 97, New York, NY 10021, USA.
Insights
Human replication factor C (hRFC) is crucial for DNA replication. Mutations in four of its five subunits disrupt ATP hydrolysis, impairing DNA synthesis and replication activity.
Area of Science:
- Molecular biology
- Biochemistry
- Cellular processes
Background:
- Human replication factor C (hRFC) is a five-subunit complex essential for DNA replication.
- hRFC catalyzes the loading of proliferating cell nuclear antigen (PCNA) onto DNA.
- This process is ATP-dependent and recruits DNA polymerases for processive DNA synthesis.
Purpose of the Study:
- To identify the specific subunit(s) of hRFC responsible for ATP hydrolysis.
- To investigate the role of individual subunits in hRFC's catalytic activity.
Main Methods:
- Site-directed mutagenesis was used to introduce point mutations in putative ATP-binding sites of each hRFC subunit.
- The replication activity of the resulting mutant hRFC complexes was assessed.
- The DNA-dependent ATPase activity of hRFC and its subcomplex (p40.p37.p36) was measured.
Main Results:
- Mutations in the ATP-binding sites of p36, p37, p40, or p140 subunits significantly reduced hRFC replication and ATPase activity.
- A mutation in the p38 subunit's ATP-binding site did not affect hRFC replication activity.
- These findings pinpoint the critical role of specific subunits in hRFC's function.
Conclusions:
- hRFC's replication activity relies on ATP hydrolysis involving at least four subunits (p36, p37, p40, and p140).
- The p38 subunit does not appear to be directly involved in ATP hydrolysis for replication.
- This study elucidates the subunit-specific contributions to hRFC's essential DNA replication function.
Abstract:
Human replication factor C (hRFC) is a five-subunit protein complex (p140, p40, p38, p37, and p36) that acts to catalytically load proliferating cell nuclear antigen onto DNA, where it recruits DNA polymerase delta or epsilon to the primer terminus at the expense of ATP, leading to processive DNA synthesis. We have previously shown that a subcomplex of hRFC consisting of three subunits (p40, p37, and p36) contained DNA-dependent ATPase activity. However, it is not clear which subunit(s) hydrolyzes ATP, as all five subunits include potential ATP binding sites. In this report, we introduced point mutations in the putative ATP-binding sequences of each hRFC subunit and examined the properties of the resulting mutant hRFC complex and the ATPase activity of the hRFC or the p40.p37.p36 complex. A mutation in any one of the ATP binding sites of the p36, p37, p40, or p140 subunits markedly reduced replication activity of the hRFC complex and the ATPase activity of the hRFC or the p40.p37.p36 complex. A mutation in the ATP binding site of the p38 subunit did not alter the replication activity of hRFC. These findings indicate that the replication activity of hRFC is dependent on efficient ATP hydrolysis contributed to by the action of four hRFC subunits.