Direct Control of the human CMG Helicase by Myc and Rb

摘要 复制的CMG(Cdc45-MCM-gins)解旋酶在通过G1-S过程中起着功能作用 S期的转换、dna复制和从复制胁迫中恢复。有证据表明，错误的- 通过致癌途径调节人CMG(HCMG)解旋酶可促进 通过失去对细胞周期的控制和造成DNA损伤而导致癌症。尽管有知识 关于细胞周期中HCMG解旋酶的亚基组成和一般功能 致癌直接调控HCMG解旋酶的分子和生化机制研究进展 人们对这些途径知之甚少。我们和其他人已经证明了两种关键蛋白质参与了 肿瘤发生，Rb和Myc，能够直接调节HCMG的组装和激活 解旋酶。肿瘤抑制因子Rb通过MCM7亚基与HCMG物理结合并抑制HCMG， 我们最近发现它来自RB的一个特殊的N-末端外显子/肽，称为外显子7(EX7)，它 在家族性遗传性癌症中丢失。癌蛋白Myc，传统上被认为具有转录功能 因子，也与HCMG相互作用，并直接(独立于转录)刺激解旋酶活性，在 部分，通过促进HCMG组装所需的染色质通路。然而，Myc也会在身体上相互作用 与HCMG合作，并必须这样做，以刺激其活动。我们提供了这种相互作用发生的证据 Myc和Rb在HCMG上接触的相同MCM7的C-末端结构域之间。这支持 假设Rb和Myc在HCMG激活过程中都以其为靶点，并在解旋酶中提供对抗作用 Rb抑制和Myc刺激HCMG活性的调节，潜在地通过相同的物理相互作用 位于HCMG上的站点。Rb和Myc在控制HCMG方面的这些角色为以下问题提供了耐人寻味的新解释 它们在肿瘤发生和细胞周期控制中的相反作用。然而，Rb和Myc如何直接调节 HCMG在生化水平上的作用尚不清楚。我们建议调查这些机械性的问题 通过以下特定目的：特异性Aim1：RB或RB-EX7肽如何抑制 人绒毛膜促性腺激素解旋酶？在没有ATP的情况下，是否阻止了ATP的水解，或者抑制了HCMG的伸长 干扰？特异性AIM2：Rb对HCMG解旋酶的抑制是否来源于对Mcm10的调节 或者是完全HCMG功能所必需的Ctf4辅助因子？具体目标3：Myc如何直接刺激 HCMG活动？Myc能抵消Rb衍生的抑制作用吗？HCMG是否需要Myc Box-II(MB-II) 刺激？

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?