RB Reversibly Inhibits DNA Replication via Two Temporally Distinct Mechanisms

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.The retinoblastoma (RB) tumor suppressor is a critical negative regulator of cellular proliferation that is targeted at high frequency in human cancer (5,30,51,61,72,73). While RB has principally been considered as a regulator of G 1 phase, the importance of RB in governing DNA synthesis has become increasingly clear (14,33,35,41,52,58,60). Currently, there are two mechanisms through which RB has been postulated to inhibit replication. In the first, DNA synthesis may be influenced by the direct interaction of RB with components of the replication process. For example, recent data have suggested a direct role for RB/E2F in regulating origin function during Drosophila chorion gene amplification (10). Furthermore, under certain conditions RB has been localized to sites of DNA replication, although this remains controversial (16, 32). Additionally, RB has been demonstrated to directly interact with the DNA replication factors MCM7, DNA polymerase ␣, and replication factor C (RFC) p140 subunit (24,55,64,67). The importance of these interactions during cellular DNA replication has yet to be determined. However, these studies collectively suggest that RB could function in cis to directly inhibit DNA replication.In the second model, RB-mediated S-phase inhibition may be attributed to the active repression of requisite DNA replication factor expression. RB is known to bin...

RETRACTED ARTICLE: Cyclin-dependent kinase 4/6 activity is a critical determinant of pre-replication complex assembly

“…Consistent with previous studies from our laboratory and others, we find that inhibition of CDK2 activity does not influence the loading of MCM proteins on chromatin, nor the expression of cdt1 and cdc6, which are required for pre-RC assembly (Braden et al, 2006;Savio et al, 2006). However, inhibition of CDK2 activity does inhibit ongoing replication in S phase as determined by retention of PCNA on chromatin (Sever-Chroneos et al, 2001;Angus et al, 2004;Braden et al, 2006;Savio et al, 2006). Thus, it is becoming increasingly clear that assembly of the pre-RC is independent of CDK2 activity.…”

“…for 5 min at 4 1C and the supernatant collected. Total cell extracts were harvested as previously described (Angus et al, 2004). Lysates were resolved by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to Immobilon-P membrane (Millipore, Bedford, MA, USA).…”

“…The accumulation of cyclin E contributes to the activation of CDK2, which further phosphorylates RB and promotes S phase entry (Hwang and Clurman, 2005). As cells progress into S phase, high levels of cyclin A are involved in DNA replication control (Angus et al, 2004;Braden et al, 2006). DNA replication is a highly ordered process that ensures cells replicate their genome only once per cell cycle (Diffley, 1994;Bell and Dutta, 2002;Blow and Hodgson, 2002;Diffley and Labib, 2002;Dimitrova et al, 2002).…”

cAMP-mediated Inhibition of DNA Replication and S Phase Progression: Involvement of Rb, p21Cip1, and PCNA

“…Disruption of PCNA activity elicited by Rb is shown to depend on E2F-mediated transcriptional repression and the ability of Rb to inhibit the expression of cyclin A has been suggested to be such an event (Sever-Chroneos et al, 2001;Angus et al, 2004). As demonstrated here, the cAMP-mediated inhibition of DNA synthesis is dependent on Rb function and correlates with dissociation of PCNA from chromatin.…”

“…protein levels of several E2F target genes involved in DNA replication (Angus et al, 2004). Disruption of PCNA activity elicited by Rb is shown to depend on E2F-mediated transcriptional repression and the ability of Rb to inhibit the expression of cyclin A has been suggested to be such an event (Sever-Chroneos et al, 2001;Angus et al, 2004).…”