Molecular analysis of the function of BRCA1-TONSL complexes

描述(申请人提供)：BRCA1(B1)是一种乳腺和卵巢肿瘤抑制物。其最著名的功能是通过同源重组(HR)修复双链断裂(DSB)。它也可以在其他类型的DNA损伤修复中发挥作用(参见下文)，以及在DNA损伤检查点中发挥作用。然而，关于B1的任何所谓功能或它如何作为肿瘤抑制物，人们的深入了解有限。为了解决这个问题，我们从一个人类细胞系中提纯了含有B1的蛋白质复合体，并用质谱仪(MS)对其进行了分析。我们希望通过更好地定义B1蛋白伙伴谱系，我们可能能够更好地理解已知的B1功能，或者根据已识别的相互作用蛋白的已知功能潜在地假设新的B1功能。在这项分析中，我们反复鉴定了一种名为TONSL(又名NFKBIL2)的蛋白质是B1相互作用因子。这种蛋白质被认为在修复倒塌的复制叉子中起作用。最近，我们的团队已经证明了B1在修复停滞不前的复制叉子中的作用。鉴于这两种蛋白质的功能相似，并基于我们对B1-TONSL相互作用的初步分析，我们假设B1将TONSL招募到折叠的复制叉子上，以帮助修复这些位点上出现的DSB断裂。这项提议的四个具体目标将集中在这一假设上。具体目标1的目标将是对B1-TONSL相互作用进行结构功能分析，作为一种从遗传学上评估B1向塌陷的叉子招募TONSL以及B1-TONSL复合体参与这些结构的修复的假设的一种手段。目标2的目标将是从我们的初步数据中理解观察结果。我们发现，在紫外线损伤后，在几乎所有的S/G2细胞中，B1都定位于紫外线损伤部位。然而，在这些细胞中，TONSL与B1在这些位置上是共定位的。目标2的目标是确定为什么会出现这种情况，这可能有助于阐明B1-TONSL复合体的功能。目标3的目标是通过亲和纯化和MS寻找并在可行的情况下鉴定B1-TONSL亚复合体的其他成员。通过这样做，我们希望识别具有已知生化功能的亚基，然后评估这些性质对B1-TONSL功能的贡献，反之亦然。这种方法可以进一步阐明这个复合体在倒塌的叉子上的功能的各个方面，和/或对这个复合体的功能提出新的假设。目标4的目标是在合作者的帮助下，通过在乳腺肿瘤的全基因组序列文库中搜索相关突变来确定TONSL和/或B1-TONSL复合体的任何其他成员是否是乳腺癌抑制基因的产物。这项工作将由候选人的赞助商在一项计划中进行监督，该计划包括以实验室为基础的研究培训，并通过参加相关的科学会议而发酵。理想情况下，这一努力的成功将有助于更好地了解B1功能以及B1功能丧失如何导致乳腺癌和/或卵巢癌。

DESCRIPTION (provided by applicant): BRCA1 (B1) is a breast and ovarian tumor suppressor. Its best known function is in the repair of double strand breaks (DSB) by homologous recombination (HR). It can also function in other types of DNA damage repair (cf below), as well as in DNA damage checkpoints. However, there is limited in depth knowledge about any of the purported functions for B1or how it acts as a tumor suppressor. To address this problem, we purified B1-containing protein complexes from a human cell line and analyzed the complexes by mass spectrometry (MS). Our hope was that by better defining the B1 protein partner repertoire we might be able to better understand known B1 functions or potentially hypothesize new B1 functions based on the known functions of identified interactors. In this analysis, we repeatedly identified a protein called TONSL (aka NFKBIL2) as a B1 interactor. This protein has been suggested to function in the repair of collapsed replication forks. Recently, our group has shown that B1 functions in the repair of stalled replication forks. Given the similar suggested function for both proteins, and based on our preliminary analysis of the B1-TONSL interaction, we hypothesize that B1 recruits TONSL to collapsed replication forks to aid in the repair of DSB breaks arising at these sites. The four specific aims of this proposal wil focus on this hypothesis. The goal of specific aim 1 will be to perform a structure function analysis on the B1-TONSL interaction as a means of assessing, genetically, the hypothesis that B1 recruits TONSL to collapsed forks and that the B1- TONSL complex is involved in repair of these structures. The goal of aim 2 will be to understand an observation from our preliminary data. We find that after UV damage, B1 localizes at UV damage sites in virtually all S/G2 cells. However, TONSL co-localizes with B1 at these sites in only some of these cells. The goal of aim 2 is to determine why this is the case, which could shed light on how B1-TONSL complexes function. The goal of aim 3 is to search for and, if feasible, identify other members of the B1-TONSL subcomplex, by affinity purification and MS. By doing this we hope to identify subunits with known biochemical functions and then assess the contributions of these properties to B1-TONSL function and vice versa. This approach could further illuminate aspects of the function of this complex at collapsed forks and/or suggest new hypotheses for the function of this complex. The goal of aim 4 is to determine, with help from collaborators, whether TONSL and/or any other member of the B1-TONSL complex is a product of a breast cancer suppressor gene by searching for relevant mutations in whole genome sequence libraries of breast tumors. This work will be supervised by the candidate's sponsor in a program composed of laboratory-based research training leavened by attendance at relevant scientific meetings. Ideally, success in this effort will contribute to a better understanding of B1 function and how loss of B1 function leads to breast and/or ovarian cancer.