Negative Regulation of DNA Replication by the Retinoblastoma Protein Is Mediated by Its Association with MCM7

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.

ACKNOWLEDGMENTS

We are indebted to members of the Horowitz laboratory for helpful discussions and to Dennis J. Templeton, Douglass Forbes, Nicholas Dyson, and Steven J. Elledge for reagents used in this study.

This work was supported in part by National Cancer Institute grant CA53248 and Faculty Research award A-73970 from the American Cancer Society to J.M.H. J.M.H. is a member of the Pew Scholars Program in the Biomedical Sciences, and J.M.S. is a fellow of the Robert Steel Foundation for Pediatric Cancer Research.