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Molecular and Cellular Biology, June 2004, p. 5404-5420, Vol. 24, No. 12
0270-7306/04/$08.00+0     DOI: 10.1128/MCB.24.12.5404-5420.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

RB Reversibly Inhibits DNA Replication via Two Temporally Distinct Mechanisms

Steven P. Angus,1,{dagger} Christopher N. Mayhew,1 David A. Solomon,1 Wesley A. Braden,1 Michael P. Markey,1 Yukiko Okuno,2 M. Cristina Cardoso,3 David M. Gilbert,2 and Erik S. Knudsen1*

Department of Cell Biology, University of Cincinnati College of Medicine, Cincinnati, Ohio 45267,1 Department of Biochemistry and Molecular Biology, SUNY Upstate Medical University, Syracuse, New York 13210,2 Max Delbrück Center for Molecular Medicine, Franz-Volhard-Klinik, D-13125 Berlin, Germany3

Received 2 October 2003/ Returned for modification 25 November 2003/ Accepted 22 March 2004

The retinoblastoma (RB) tumor suppressor is a critical negative regulator of cellular proliferation. Repression of E2F-dependent transcription has been implicated as the mechanism through which RB inhibits cell cycle progression. However, recent data have suggested that the direct interaction of RB with replication factors or sites of DNA synthesis may contribute to its ability to inhibit S phase. Here we show that RB does not exert a cis-acting effect on DNA replication. Furthermore, the localization of RB was distinct from replication foci in proliferating cells. While RB activation strongly attenuated the RNA levels of multiple replication factors, their protein expression was not diminished coincident with cell cycle arrest. During the first 24 h of RB activation, components of the prereplication complex, initiation factors, and the clamp loader complex (replication factor C) remained tethered to chromatin. In contrast, the association of PCNA and downstream components of the processive replication machinery was specifically disrupted. This signaling from RB occurred in a manner dependent on E2F-mediated transcriptional repression. Following long-term activation of RB, we observed the attenuation of multiple replication factors, the complete cessation of DNA synthesis, and impaired replicative capacity in vitro. Therefore, functional distinctions exist between the "chronic" RB-mediated arrest state and the "acute" arrest state. Strikingly, attenuation of RB activity reversed both acute and chronic replication blocks. Thus, continued RB action is required for the maintenance of two kinetically and functionally distinct modes of replication inhibition.

* Corresponding author. Mailing address: Department of Cell Biology, Vontz Center for Molecular Medicine, 3125 Eden Ave., Cincinnati, OH 45267-0521. Phone: (513) 558-8885. Fax: (513) 558-2445. E-mail: Erik.Knudsen{at}uc.edu.

{dagger} Present address: Department of Molecular Genetics and Microbiology, Howard Hughes Medical Institute, Duke University Medical Center, Durham, NC 22710.

Molecular and Cellular Biology, June 2004, p. 5404-5420, Vol. 24, No. 12
0022-538X/04/$08.00+0     DOI: 10.1128/MCB.24.12.5404-5420.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.



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