The N Terminus of the Retinoblastoma Protein Inhibits DNA Replication via a Bipartite Mechanism Disrupted in Partially Penetrant Retinoblastomas

Abstract

The N-terminal domain of the retinoblastoma (Rb) tumor suppressor protein (RbN) harbors in-frame exon deletions in partially penetrant hereditary retinoblastomas and is known to impair cell growth and tumorigenesis. However, how such RbN deletions contribute to Rb tumor- and growth-suppressive functions is unknown. Here we establish that RbN directly inhibits DNA replication initiation and elongation using a bipartite mechanism involving N-terminal exons lost in cancer. Specifically, Rb exon 7 is necessary and sufficient to target and inhibit the replicative CMG helicase, resulting in the accumulation of inactive CMGs on chromatin. An independent N-terminal loop domain, which forms a projection, specifically blocks DNA polymerase α (Pol-α) and Ctf4 recruitment without affecting DNA polymerases ε and δ or the CMG helicase. Individual disruption of exon 7 or the projection in RbN or Rb, as occurs in inherited cancers, partially impairs the ability of Rb/RbN to inhibit DNA replication and block G₁-to-S cell cycle transit. However, their combined loss abolishes these functions of Rb. Thus, Rb growth-suppressive functions include its ability to block replicative complexes via bipartite, independent, and additive N-terminal domains. The partial loss of replication, CMG, or Pol-α control provides a potential molecular explanation for how N-terminal Rb loss-of-function deletions contribute to the etiology of partially penetrant retinoblastomas.

ACKNOWLEDGMENTS

We thank Johannes Walter, Julian Blow, Rolf Knippers, William Dunphy, Karlene Cimprich, Anindya Dutta, Anna Philpott, and Shou Waga for sharing valuable antibody reagents and John L. Cleveland for editing and critical review of the manuscript.

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication. We state that there is no conflict of interest regarding the data presented in the paper.

Additional information

Funding

The Alexandrow laboratory was supported by NIH grants R01-CA130865 and R21-CA155393 and by NCI Comprehensive Cancer Center grant P30-CA076292, awarded to the H. Lee Moffitt Cancer Center and Research Institute.