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== Cdc6 is phosphorylated by CycE-Cdk2, which is required for MCM loading (31). phase by inhibiting pre-RC activation and suggest a novel role for Rb in mediating this effect of TGF-1 through direct interaction with and control of the MCM helicase. Transforming growth factor 1 (TGF-1) Meropenem is a potent inhibitor of cell proliferation. TGF-1-induced arrest occurs during G1and is mediated Meropenem by Smad proteins, which regulate transcriptional targets, including c-myc(11,13,37). Downregulation of c-mycallows induction of the Cdk inhibitor (CKI) p15INK4B, which inhibits Cdk4-CycD (20,45). The p27Kip1inhibitor is also utilized by TGF-1 to inhibit Cdk2-CycE (39). Cdk suppression prevents hyperphosphorylation of Rb (28), causing Rb to remain in a hypophosphorylated, growth-suppressive form. The pivotal roles for c-mycsuppression and Rb are illustrated by the demonstration that ectopic c-Myc or viral tumor proteins that inactivate Rb override TGF-1 (3,28,38). However, this pathway utilized by TGF-1 has been largely derived from experimentation in which TGF-1 is added to cells in early G1, prior to the occurrence of most G1events, and progression into late-G1/S phase is hindered due to these mechanisms. Several studies have raised important questions with regard to the mechanisms of TGF-1 signaling. Cells lacking p27Kip1, p15INK4B, or p21Cip1remain sensitive to Meropenem growth arrest by TGF-1 (24,34,46). Thus, CKIs are not absolutely required for TGF-1 to arrest cells, and it has been suggested that transcriptional suppression of Cdc25A is an alternative means for TGF-1 to suppress Cdks (24). TGF-1 can block S-phase entry when added to cells in early G1or late G1, including just prior to G1/S, after most G1events have already occurred (4,23). This ability of late-G1TGF-1 exposure to acutely block G1-S transit is Meropenem particularly intriguing, since mammalian cells no longer require de novo mRNA synthesis in late G1for S-phase entry (4,9,33) and the effectiveness of TGF-1 arrest after exposure in late G1is not affected by agents that block de novo mRNA synthesis (4,23). Thus, TGF-1 signals in late G1acutely block S-phase entry utilizing mechanisms independent of transcriptional upregulation or downregulation. This calls into question the need for acute transcriptional control of c-Myc, Cdc25A, CKIs, or other transcriptional targets by TGF-1 specifically in late G1and elicits questions as to the transcription-independent and acute nature of the mechanisms by which TGF-1 achieves arrest when added to cells in late G1. We reasoned that TGF-1 likely produces negative effects on the prereplication complex (pre-RC) and that understanding such effects might offer insight into TGF-1 signaling that explains these unanswered questions. Pre-RCs assemble at future origins of DNA replication and play a pivotal role in regulating the transition to S phase (6). Each pre-RC is composed of the origin recognition complex (ORC), which recruits Cdt1, Cdc6, and the hexameric minichromosome maintenance (MCM) helicase. Initiation of DNA replication (i.e., the G1/S transition) commences after Cdc45, DNA polymerases, and PCNA (and other proteins) are recruited to the pre-RCs and MCMs are activated to melt origin DNA (6). We show here that TGF-1 signals do indeed target pre-RC functionality and that the effects of TGF-1 on pre-RC dynamics provide Meropenem novel explanations for these unanswered questions. TGF-1 treatment in early G1, prior to pre-RC assembly, blocks such assembly and causes numerous other cell cycle changes, including suppression of Myc and inhibition of CycE-Cdk2. In contrast, treatment with TGF-1 in late G1, after pre-RCs have already assembled, does not cause disassembly of pre-RCs. Instead, TGF-1 acutely inhibits pre-RC activation and arrests cells prior to the helicase unwinding step at G1/S. This late-G1arrest does not involve CycE-Cdk2 inhibition or Myc suppression. However, Rb is critically required for acute inhibition of pre-RC activation by TGF-1. Rb mediates TGF-1 arrest in late G1via direct targeting of the MCM helicase, specifically through Mcm7. Loss of FLJ12788 Rb or gain of Mcm7 overrides TGF-1 arrest in late G1, indicating that the Rb-MCM interaction plays a pivotal role. These observations provide novel insight into the mechanisms of TGF-1 arrest and suggest a role for Rb in mediating late-G1arrest by TGF-1 that involves regulating the MCM complex. == MATERIALS AND METHODS == == Cell culture, synchronization, and drugs. == Mouse keratinocytes (BALB/MK) were maintained as described previously (3). HaCaT and SaOS-2.