Direct Control of the human CMG Helicase by Myc and Rb

Myc 和 Rb 直接控制人 CMG 解旋酶

基本信息

批准号：
10624906
负责人：
Mark G. Alexandrow
金额：
$ 32.94万
依托单位：
H. LEE MOFFITT CANCER CTR & RES INST
依托单位国家：
美国
项目类别：
财政年份：
2021
资助国家：
美国
起止时间：
2021-06-01 至 2025-05-31
项目状态：
未结题

项目摘要

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?

抽象的通过G1-S的通过 S相过程中的复制应力中的过渡，DNA复制和恢复。有证据表明错误通过致癌途径调节人CMG（HCMG）解旋酶可以有助于发展通过失去细胞周期控制和DNA损伤的产生而癌症。虽然有知识关于细胞周期中HCMG解旋酶的亚基组成和一般功能直接通过致致致致致癌酶调节HCMG解旋酶的进展，分子和生化机制途径知之甚少。我们和其他人表明，两个关键蛋白涉及肿瘤发生RB和MYC能够直接调节HCMG的组装和激活解旋酶。肿瘤抑制器RB通过MCM7亚基与HCMG物理结合，并抑制HCMG，我们最近显示的是源自RB中的特定N末端外显子/肽，称为外显子7（EX7）在家族遗传的癌症中迷失了。传统上被认为是转录的癌蛋白MYC 因子，也与HCMG相互作用，直接（独立于转录）刺激了解旋酶活性，在部分，通过促进HCMG组装所需的染色质访问。但是，MYC也有身体互动使用HCMG，必须这样做才能刺激其活性。我们提供了这种互动发生的证据 MYC与RB在HCMG上接触的MCM7的同一C端域之间。这支持 RB和MYC在激活过程中均针对HCMG的假设，并在解旋酶中提供反作用调节RB抑制和MYC刺激HCMG活性，可能通过相同的物理相互作用 HCMG上的站点。 RB和MYC在控制HCMG方面的这种角色为您提供了有趣的小说解释它们在肿瘤发生和细胞周期控制中的相对作用。但是，RB和MYC如何直接调节生化水平的HCMG仍然未知。我们建议调查这些机械问题通过以下特定目的：特定目标1：RB或RB-EX7肽如何抑制活性在体外纯化的HCMG解旋酶？被ATP水解阻断，或者没有ATP抑制HCMG延伸干涉？特定AIM2：RB对RB的抑制作用抑制了MCM10的调节还是CTF4全HCMG功能所需的CTF4副因素？特定AIM3：MYC如何直接刺激 HCMG活动？ MYC是否应对RB衍生的抑制作用？ HCMG所需的Myc Box-II（MB-II）刺激？