Analysis of regulation and function of the BLM helicase

描述(由申请人提供)：基因组不稳定是一种 突变发生频率高于正常的细胞。这样的一个 这种情况可能是由于DNA复制、DNA修复或细胞的 损害控制机器。这些细胞系统中的一个的主要缺陷 是一种罕见的常染色体隐性遗传病--布卢姆综合征(BS)。这个 BS基因BLm编码一个RecQ DNA解旋酶。BLM解旋酶不存在于 细胞导致显著的基因组不稳定性，包括两条染色体 断裂和过度染色单体交换，尤其是姐妹染色单体 交换(SCE)。的超可变和超可重组特性 BS细胞无疑解释了BS患者显著的癌症易感性； 然而，博莱姆解旋酶的正常功能(S)和分子 蛋白质缺失导致BS基因组不稳定的机制 细胞是不被理解的。 博莱姆解旋酶以弥漫的、微斑点的形式存在于细胞中，并以 我们已经确定为PML核体的特征核点 (PML-NBS)。和PML一样，BLM被泛素类分子共价修饰， SUMO-1和SUMO-2，我们假设它们的功能是将BLM本地化到 PML-NBS。该提案的目标1是确定和描述以下区域的特征 调解PML-NBS本地化的BLM。我们在实验中引入了 不同的突变(缺失、插入和氨基酸替换) GFP-BLM表达载体的构建及在不同环境中的表达 细胞，包括HeLa和BS细胞。然后，我们分析功能后果 对PML-NBS、BLM解旋酶活性的定位突变，以及 姐妹染色单体交换试验对BS细胞表型的补充。目标2是 确定BLM的相扑修改是否导致本地化到PML-NBS 通过执行类似于AIM I中的突变分析并使用In 体外修饰实验绘制相互作用和相扑位点图。我们分析了 修饰对BLM定位、解旋酶活性、 BS细胞表型的互补性。在目标3中，我们调查了 博莱姆-拓扑异构酶IIIα(TOPO III)复合体的功能。我们表演了一场 与Topo III相互作用的BLM结构域的突变分析 酵母双杂交筛选。然后，我们确定了函数的后件 这些突变通过测量蛋白质相互作用的能力， 该复合体在解旋酶和拓扑异构酶测定中的活性及其在 B细胞致密化。此外，我们还测量了相扑的效果 BLM Topo III相互作用的修饰及配合物的酶活性。 在目标4中，我们分析了BLM的域结构以及BLM在其中的作用 解旋酶活性的寡聚和BS细胞的互补。

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.