RB reversibly inhibits DNA replication via two temporally distinct mechanisms
Authors: Christopher N Mayhew
Steven P Angus
Erik S Knudsen
Wesley A Braden
Yukiko Okuno
Michael P Markey
David M Gilbert
M Cristina Cardoso
David A Solomon
Journal: Molecular and cellular biology (2004)
Discipline: MOLECULAR BIOLOGY & GENETICS
Keywords: retinoblastoma; tumor suppressor; cellular proliferation; E2F-dependent transcription; DNA replication
Affiliations: Department of Cell Biology, University of Cincinnati College of Medicine, OH 45267, USA., Department of Cell Biology, University of Cincinnati College of Medicine, Cincinnati, Ohio 45267, Department of Biochemistry and Molecular Biology, SUNY Upstate Medical University, Syracuse, New York 13210, Max Delbrück Center for Molecular Medicine, Franz-Volhard-Klinik, D-13125 Berlin, Germany

Abstract:  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. 

Retracted: No

PubPeer comments:
 Figure 4D. Much more similar than you wuld expect. https: imgur.com FSOxqRS , 
 Figure 9B. PCNA and MCM7 bands much more similar than you would expect given quite different protein molecular weights https: imgur.com g5L1gM3 , 
 Let me reach out to the primary author for this paper. A2-4 are a derivative of Rat1 cells, but re-using the same blots is very poor judgement. Since both papers are at Molecular Cellular Biology, I will reach out to the editors for correction , 
 #3 Mol Cell Biol. 2006 Oct;26(20):7667-81 figure 1C. There appears to be a transduction with GFP in the left lane. Legend. (C) A2-4 cells cultured in the presence of DOX were transduced with GFP- or p16ink4a (p16)-encoding adenoviruses and harvested at 24 h postinfection. Left panel: cells were harvested, equal total protein amounts were resolved by SDS-PAGE, and the indicated proteins were detected by immunoblotting. Right panel: cells were harvested, fixed, stained with propidium iodide, and processed for flow cytometry. Histograms are from 10,000 events. Mol Cell Biol. 2004 Jun;24(12):5404-20. Legend. Figure 4D. (D, left panel) Asynchronously proliferating Rat-1 cells were infected with recombinant adenovirus expressing p16ink4a. Cells were harvested at 0 (lane 1) or 24 (lane 2) h postinfection. Equal total protein levels were resolved by SDS-PAGE, and the indicated proteins were detected by immunoblotting. The image is of the left panel. There does not seem to be any transduction with GFP. The right panel does refer to GFP. (Right panel) Rat-1 cells were infected with GFP- or p16ink4a-encoding adenoviruses and subjected to in situ extraction with CSK buffer at 24 h postinfection. Cells were subsequently fixed, and PCNA was detected by immunofluorescence. The values shown are means u00b1 standard deviation. At least 200 cells were counted per experiment. ,