Identification/characterization Complex-Bloom Syndrome

布卢姆综合征是一种罕见的人类遗传性疾病，患者表现为生长迟缓、免疫缺陷、不孕不育、光敏和易患癌症。BS中的缺陷基因最近被克隆(命名为BLM)，并被发现属于一个进化保守的解旋酶家族，称为RecQ。重组的BLMp蛋白在体外具有解旋酶活性，这表明BS可能是由DNA代谢反应的缺陷引起的，如复制或修复。有趣的是，BLM基因属于解旋酶家族，与Werner综合征和Rothmund-Thomson综合征(RTS)中突变的基因一样。这三种疾病都有一些共同的特征，比如遗传不稳定和易患癌症。但每种疾病都有其独特的症状。例如，WS患者过早地表现出许多与年龄相关的特征，包括骨质疏松症、动脉粥样硬化、糖尿病和白内障，这些在BS或RTS中没有观察到。此外，WS患者不像BS患者那样表现出免疫缺陷或光敏。为了了解这些人类疾病的分子机制，我们建议分离包含每个基因产物的蛋白质复合体。为了探讨BS的作用机制，我们从人HeLa中提取了三个含有BS、BLM中缺失的解旋酶的复合体。有趣的是，其中一个被称为BRAFT的复合体还包含五个Fanconi贫血(FA)互补组蛋白(FA)。FA在基因组不稳定性和癌症易感性方面与BS相似，但其大部分基因产物没有已知的生化活性，其发病机制尚不清楚。BRAFT显示出DNA解旋活性，这需要BLM的存在，因为从BLM缺陷的细胞中分离出来的复合体缺乏这种活性。该复合体还包含拓扑异构酶IIIa和复制蛋白A，这两种蛋白质已知与BLM相互作用，可能有助于DNA的解离。我们发现从FA细胞系分离的BLM复合体具有较低的分子质量。我们的研究首次提供了多蛋白FA复合体的生化特征，并暗示了基因组维持的BLM和FA途径之间的联系。FA蛋白是DNA解离复合体的一部分，这一发现暗示FA蛋白可能参与DNA修复。这项研究的大部分最近都发表在《摩尔》杂志上。牢房。比奥尔。 在今年发表在EMBO J上的一篇论文中，我们表明BLAP75是来自HeLa细胞的所有三个BLM复合体的组成部分。利用siRNA敲除技术，我们证明了BLAP75对于体内BLM复合体的稳定性是必不可少的。BLAP75在BLM介导的过程中起着一致的作用，作为对DNA损伤的反应，BLAP75与BLM共定位于核下病灶，其缺失会损害BLM向这些病灶的募集。BLAP75被siRNA耗尽也会导致有丝分裂期间BLM的磷酸化不足，以及细胞增殖缺陷。此外，去除BLAP75的细胞表现出姐妹染色单体交换水平的增加，类似于被siRNA去除的BLM的细胞。因此，BLAP75是维持基因组完整性的BLM相关细胞机制的重要组成部分。在我们的论文发表后，另外两个实验室使用遗传学方法证明了BLAP75的酵母同源基因也是RecQ解旋酶-Topo IIIa复合体的组成部分，是维持基因组稳定所必需的。因此，人类的生物化学和酵母的遗传学都得出了相同的结论。综上所述，这些数据表明BLAP75及其同源物在不同物种中具有保守的基因组保护功能。 我们目前正在研究另一种BLM复合体成分BLAP250在基因组维持中的重要性。

Bloom syndrome (BS) is a rare human genetic disease in which patients exhibit growth retardation, immunodeficiency, infertility, photosensitivity, and predisposition to cancer. The gene defective in BS has recently been cloned (named BLM) and was found to belong to an evolutionarily conserved helicase family, called RecQ. The recombinant BLMp protein has been shown to possess a helicase activity in vitro, suggesting that BS could be caused by a defect in a DNA metabolic reaction, such as replication or repair. Interestingly, BLM gene belongs to the helicase family, like the genes mutated in Werner Syndrome and Rothmund-Thomson syndrome (RTS). All three diseases have some common features, such as genetic instability and predisposition to cancer. But each disease has its own distinctive symptoms. For example, WS patients prematurely display many age-related features, including osteoporosis, atherosclerosis, diabetes and cataracts, which are not observed in BS or RTS. Also, WS individuals do not show immunodeficiency or photosensivity like BS patients. To understand the molecular mechanism of these human diseases, we propose to isolate the protein complexes containing each gene product. To investigate the mechanism of BS, we isolated from human HeLa extracts three complexes containing the helicase defective in BS, BLM. Interestingly, one of the complexes, termed BRAFT, also contains five of the Fanconi anemia (FA) complementation group proteins (FA). FA resembles BS in genomic instability and cancer predisposition, but most of its gene products have no known biochemical activity and the molecular pathogenesis of the disease is poorly understood. BRAFT displays a DNA-unwinding activity, which requires the presence of BLM because complexes isolated from BLM-deficient cells lack such an activity. The complex also contains topoisomerase IIIa and replication protein A, proteins that are known to interact with BLM and could facilitate unwinding of DNA. We find that BLM complexes isolated from a FA cell line have a lower molecular mass. Our study provides the first biochemical characterization of a multiprotein FA complex and suggests a connection between the BLM and FA pathways of genomic maintenance. The findings that FA proteins are part of a DNA-unwinding complex imply that FA proteins may participate in DNA repair. Much of this work has recently been published in Mol. Cell. Biol. In a paper published in EMBO J this year, we showed that BLAP75 is a component of all three BLM complexes from HeLa cells. Using siRNA knockdown techniques, we show that BLAP75 is essential for BLM complex stability in vivo. Consistent with a role in BLM-mediated processes, BLAP75 co-localizes with BLM in subnuclear foci in response to DNA damage, and its depletion impairs the recruitment of BLM to these foci. Depletion of BLAP75 by siRNA also results in deficient phosphorylation of BLM during mitosis, as well as defective cell proliferation. Moreover, cells depleted of BLAP75 display an increased level of sister-chromatid exchange, similar to cells depleted of BLM by siRNA. Thus, BLAP75 is an essential component of the BLM-associated cellular machinery that maintains genome integrity. After our paper has been published, two other labs used genetic approaches to show that the yeast homolog of BLAP75 is also a component of RecQ helicase-Topo IIIa complex, and is required for maintaining genome stability. Thus, both biochemistry in human and genetics in yeast have reached the same conclusion. Together, these data suggest that BLAP75 and its homologs in various species have a conserved function in guarding genome. We are currently examining the importance of another BLM complex component, BLAP250, in genome maintanence.