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Negative regulation of DNA replication by the retinoblastoma protein is mediated by its association with MCM7
J M Sterner1, S Dew-Knight, C Musahl
1Department of Molecular Cancer Biology, Duke University Medical Center, Durham, North Carolina 27710, USA.
Abstract:
A yeast two-hybrid screen was employed to identify human proteins that specifically bind the amino-terminal 400 amino acids of the retinoblastoma (Rb) protein. Two independent cDNAs resulting from this screen were found to encode the carboxy-terminal 137 amino acids of MCM7, a member of a family of proteins that comprise replication licensing factor. Full-length Rb and MCM7 form protein complexes in vitro, and the amino termini of two Rb-related proteins, p107 and p130, also bind MCM7. Protein complexes between Rb and MCM7 were also detected in anti-Rb immunoprecipitates prepared from human cells. The amino-termini of Rb and p130 strongly inhibited DNA replication in an MCM7-dependent fashion in a Xenopus in vitro DNA replication assay system. These data provide the first evidence that Rb and Rb-related proteins can directly regulate DNA replication and that components of licensing factor are targets of the products of tumor suppressor genes.
Insights
Retinoblastoma (Rb) protein binds MCM7, a component of DNA replication licensing factor. This interaction directly regulates DNA replication, implicating tumor suppressor proteins in controlling this crucial cellular process.
Area of Science:
- Molecular Biology
- Cell Biology
- Biochemistry
Background:
- The retinoblastoma (Rb) protein is a well-known tumor suppressor.
- DNA replication licensing factors are essential for controlling DNA replication initiation.
- The interaction between tumor suppressors and replication machinery is not fully understood.
Purpose of the Study:
- To identify proteins that bind the retinoblastoma (Rb) protein.
- To investigate the functional relationship between Rb and DNA replication components.
Main Methods:
- Yeast two-hybrid screening to identify binding partners of Rb.
- In vitro protein complex formation assays.
- Immunoprecipitation from human cell extracts.
- Xenopus in vitro DNA replication assays.
Main Results:
- Identified MCM7, a component of replication licensing factor, as an Rb-binding protein.
- Demonstrated direct binding between Rb and MCM7 in vitro and in human cells.
- Showed that Rb and related proteins (p107, p130) inhibit DNA replication in an MCM7-dependent manner.
Conclusions:
- Rb and Rb-related proteins directly regulate DNA replication.
- Components of the DNA replication licensing factor are targets of tumor suppressor proteins.
- Establishes a novel mechanism for tumor suppressor-mediated control of DNA replication.