Analysis of regulation and function of the BLM helicase

描述（由申请人提供）：基因组不稳定性是一种 突变发生频率高于正常的细胞。这样的 这种情况可能是由于DNA复制，DNA修复或细胞的缺陷引起的。 损害控制机械这些细胞系统中的一个主要缺陷 罕见的常染色体隐性遗传疾病Bloom综合征（BS）的特征。的 BS基因BLM编码RecQ DNA解旋酶。BLM解旋酶的缺失来自 细胞会引起显著的基因组不稳定性， 断裂和过度染色单体交换，特别是姐妹染色单体 交换（SCE）。超突变性和超重组性特征 BS细胞无疑解释了BS人显著的癌症倾向; 然而，BLM解旋酶的正常功能和分子的 蛋白质的缺失导致BS中基因组不稳定的机制 细胞不被理解。 BLM解旋酶在细胞中以弥散的、微斑点的形式存在， 我们已经确定为PML核体的特征核点 （PML-NBs）。与PML一样，BLM被泛素样分子共价修饰， SUMO-1和SUMO-2，我们假设它们的功能是将BLM定位于 PML-NB。该提案的目标1是确定和描述 BLM介导定位到PML-NB。我们实验性地引入 不同的突变（缺失，插入和氨基酸取代）， GFP-BLM表达构建体，并在各种细胞中表达突变蛋白。 细胞，包括HeLa和BS细胞。然后，我们分析功能的后果， 定位于PML-NB的突变，BLM解旋酶活性，和 通过SCE测定法测定BS细胞表型的互补。目标二是 确定BLM的SUMO修饰是否导致PML-NB的定位 通过进行类似于Aim I中的突变分析并使用in 体外修饰测定以绘制相互作用和SUMO位点。我们分析 修饰对BLM定位，解旋酶活性， 和BS细胞表型的互补。在目标3中，我们研究了 BLM-拓扑异构酶III α（Topo III）复合物的功能。我们进行了 BLM与拓扑异构酶III相互作用的结构域的突变分析， 酵母双杂交筛选然后，我们确定的功能后果 通过测量蛋白质相互作用的能力来测量这些突变， 复合物在解旋酶和拓扑异构酶测定中的活性，以及其在 BS细胞补偿。此外，我们还测量了相扑的效果， 对BLM Topo III相互作用和复合物酶活性的修饰。 在目标4中，我们分析了BLM的结构域结构和BLM的作用。 解旋酶活性和BS细胞互补中的寡聚化。

DESCRIPTION (provided by applicant): Genomic instability is a condition of a cell in which mutations occur at a frequency greater than normal. Such a condition can arise from defects in DNA replication, DNA repair, or the cell's damage control machinery. A primary defect in one of these cellular systems characterizes the rare autosomal recessive disorder Bloom syndrome's (BS). The BS gene, BLM, encodes a RecQ DNA helicase. Absence of the BLM helicase from a cell causes a striking genomic instability that includes both chromosome breakage and excessive chromatid exchange, in particular, sister chromatid exchange (SCE). The hypermutability and hyperrecombinability characteristic of BS cells doubtless explains the striking cancer predisposition of BS persons; however, the normal function(s) of the BLM helicase and the molecular mechanisms by which the protein's absence causes genomic instability in BS cells are not understood. The BLM helicase is present in the cell in a diffuse, microspeckled form and in characteristic nuclear dots that we have identified as the PML nuclear bodies (PML-NBs). Like PML, BLM is covalently modified by a ubiquitin like molecule, SUMO-1 and SUMO-2, which we hypothesize functions to localize BLM to the PML-NBs. Aim 1 of the proposal is to identify and characterize the regions of BLM that mediate localization to the PML-NBs. We experimentally introduce different mutations (deletions, insertions, and amino acid substitutions) into a GFP-BLM expression construct and express the mutant proteins in various cells, including HeLa and BS cells. Then we assay the functional consequences of the mutations on localization to the PML-NBs, BLM helicase activity,and complementation of the BS cellular phenotype by the SCE assay. Aim 2 is to determine whether SUMO modification of BLM causes localization to the PML-NBs by performing a mutational analysis similar to that in Aim I and using an in vitro modification assay to map interactions and SUMO-sites. We analyze the functional consequences of modification on BLM localization, helicase activity, and complementation of the BS cellular phenotype. In Aim 3, we investigate the function of the BLM-Topoisomerase III alpha (Topo III) complex. We perform a mutational analysis of the domain of BLM that interacts with Topo III by the yeast two-hybrid screen. Then, we determine the functional consequences of these mutations by measuring the ability of the proteins to interact, the activity of the complex in helicase and topoisomerase assays, and its action in BS cell compimentation. Additionally, we measure the effect of SUMO modification on BLM Topo III interaction and the complexes enzymatic activity. In Aim 4, we analyze the domain structure of BLM and the role of oligomerization in helicase activity and BS cell complementation.