Role of Bub1 in SV40 large T antigen mediated transformation and replication

描述（申请人提供）：SV 40大T抗原（LT）是研究DNA复制和恶性转化的有力模型。我们已经确定了LT与有丝分裂调节Bub1的相互作用。Bub1对于保持基因组完整性至关重要。它主要作用于纺锤体检查点，在那里它在监测姐妹染色单体着丝粒的张力和附着中起关键作用。Bub1在某些以非整倍体为特征的人类癌症中发生突变。LT与Bub1的结合导致纺锤体检查点的衰减。我们假设Bub1与LT的结合解释了LT使宿主基因组不稳定的能力，特别是诱导四倍体的能力。遗传分析表明，LT与Bub1的结合不仅是致癌转化所必需的，也是病毒DNA复制所必需的。我们的分析进一步表明，LT与Bub1的结合与p53稳定性有关，这可能是DNA损伤反应的结果。我们的观察结果与LT作为分子支架指导Bub1磷酸化靶点（如p53）的模型一致。与Bub1的相互作用表明LT的新作用，可能揭示它如何导致染色体畸变，p53稳定和转化。我们提出以下目的来详细研究这种相互作用：1.从机制上确定LT如何通过Bub1进行转换，以及函数2的p53增益的潜在贡献。表征LT如何使用Bub1诱导四倍体和DNA损伤反应3.阐明LT如何通过Bub1结合促进病毒复制。公共卫生相关性：对SV40 DNA肿瘤病毒的大T抗原的研究为肿瘤诱导和DNA分子的精确复制（复制）所需的基本机制提供了重要的见解。关键的细胞靶点，如p53肿瘤抑制因子，首先使用该模型确定。我们的特点是一种新的结合伙伴的T抗原称为Bub1。有趣的是，Bub1密切监测和保护细胞分裂，长期以来一直怀疑T抗原破坏了这一过程，从而导致染色体异常。在某些人类癌症中发现了Bub1突变，这些癌症也以染色体异常为特征。我们的建议将调查如何机械T抗原结合到Bub1所需的恶性转化和复制。在这个过程中，我们希望更好地了解正常的Bub1功能，以及它的妥协如何促进肿瘤发生。

DESCRIPTION (provided by applicant): SV40 large T antigen (LT) is a powerful model to study DNA replication and malignant transformation. We have identified an interaction of LT with the mitotic regulator Bub1. Bub1 is critical for preserving genome integrity. It primarily acts at the spindle checkpoint, where it serves a critical function in monitoring tension and attachment at sister chromatid kinetochores. Bub1 is mutated in certain human cancers characterized by aneuploidy. LT binding to Bub1 leads to attenuation of the spindle checkpoint. We hypothesize that binding of Bub1 to LT accounts for some of LT's ability to destabilize the host genome, in particular the ability to induce tetraploidy. Genetic analysis shows that LT binding to Bub1 is required not only for oncogenic transformation but also for viral DNA replication. Our analysis further demonstrates that LT binding to Bub1 is linked to p53 stabilization, which is likely a consequence of a DNA damage response. Our observations are consistent with a model whereby LT acts as a molecular scaffold to direct Bub1 to phosphorylate targets such as p53. The interaction with Bub1 suggests novel roles for LT that may shed light on how it causes chromosome aberrations, stabilization of p53 and transformation. We propose the following aims to study the interaction in detail: 1. Determine mechanistically how LT transforms via Bub1 and the potential contribution of a p53 gain of function 2. Characterize how LT uses Bub1 to induce tetraploidy and DNA damage responses 3. Elucidate how LT promotes viral replication via Bub1 binding. PUBLIC HEALTH RELEVANCE: Studies of the large T antigen of the SV40 DNA tumor virus have provided significant insight on basic mechanisms required for tumor induction and accurate duplication of DNA molecules (replication). Critical cellular targets such as the p53 tumor suppressor were first identified using this model. We have characterized a novel binding partner for T antigen called Bub1. Interestingly, Bub1 closely monitors and safeguards cell division, a process that T antigen has long been suspected to disrupt and thereby cause chromosomal abnormalities. Bub1 mutations are found in certain human cancers also characterized by chromosomal abnormalities. Our proposal will investigate how mechanistically T antigen binding to Bub1 is required for malignant conversion and replication. In the process, we expect to gain a better understanding of normal Bub1 function, and how its compromise facilitates tumorigenesis.