Abstract The replicative CMG (Cdc45-MCM-GINS) helicase plays functional roles during passage through the G1-S transition, DNA replication, and recovery from replicative stresses during S-phase. Evidence suggests that mis- regulation of the human CMG (hCMG) helicase by oncogenic pathways can contribute to the development of cancer through loss of cell cycle control and the creation of DNA damage. Although there is knowledge regarding the subunit composition and general functionality of the hCMG helicase during cell cycle progression, the molecular and biochemical mechanisms regulating the hCMG helicase directly by oncogenic pathways are poorly understood. We and others have shown that two pivotal proteins involved in tumorigenesis, Rb and Myc, are capable of directly regulating the assembly and activation of the hCMG helicase. The tumor suppressor Rb physically binds to the hCMG via the Mcm7 subunit and inhibits the hCMG, which we recently showed was derived from a specific N-terminal exon/peptide in Rb called Exon 7 (Ex7) that is lost in familial inherited cancers. The oncoprotein Myc, traditionally thought to function as a transcription factor, also interacts with the hCMG and directly (independent of transcription) stimulates helicase activity, in part, by promoting chromatin access necessary for hCMG assembly. However, Myc also physically interacts with the hCMG and must do so to stimulate its activity. We provide evidence that this interaction occurs between Myc and the same C-terminal domain of Mcm7 that Rb contacts on the hCMG. This supports the hypothesis that Rb and Myc both target the hCMG during its activation and provide countering roles in helicase regulation, with Rb inhibiting and Myc stimulating hCMG activity, potentially via the same physical interaction site on the hCMG. Such roles for Rb and Myc in controlling the hCMG offer intriguing novel explanations for their opposing roles in tumorigenesis and cell cycle control. However, how Rb and Myc directly regulate the hCMG at the biochemical level remains unknown. We propose to investigate these mechanistic questions through the following Specific Aims: Specific Aim1: How does Rb or Rb-Ex7 peptide inhibit the activity of the purified hCMG helicase in vitro? Is ATP hydrolysis blocked, or hCMG elongation suppressed without ATP interference? Specific Aim2: Does inhibition of the hCMG helicase by Rb derive from regulation of the Mcm10 or Ctf4 co-factors necessary for full hCMG functionality? Specific Aim3: How does Myc directly stimulate hCMG activity? Does Myc counteract Rb-derived inhibition? Is Myc Box-II (MB-II) required for hCMG stimulation?