Function of the Bloom's syndrome DNA helicase in the maintainance of genome integrity

项目摘要 RecQ样DNA解旋酶BLM因其在DNA双链反应和修复中的关键作用而闻名。 哺乳动物细胞中的链断裂。BLM活性的破坏导致Bloom综合征，其特征在于 患癌症的风险极高，身材矮小，平均寿命只有25岁。癌症易感性， 染色体断裂和其他细胞缺陷目前被解释为缺乏BLM的活动， DNA损伤反应和同源重组。在这个提议中，我们正在检验这样一个假设， BLM在DNA复制起始和延伸过程中起着重要作用，以维持染色体的稳定性 在未受干扰的细胞中。这一假设是基于广泛的初步数据，包括一个公正的筛选， 导致发现染色质结合的BLM直接与Mcm 6相互作用的中期S期蛋白质组 染色质结合Mcm 2 -7亚基。值得注意的是，BLM和Mcm 6中的两个不同的结合位点差异 调节G1和S期复合物的形成，并破坏S期BLM/Mcm 6相互作用，但 而不是在G1期，导致超正常的DNA复制速度。DNA复制速度异常加速 超过安全限度是一种导致DNA损伤和杀死某些类型癌症的机制 细胞我们的初步研究结果表明，BLM/Mcm 6相互作用作为一种新的，负调节 人类细胞中的DNA复制。缺乏BLM的细胞不会表现出更快的复制速度 表明加速复制需要BLM蛋白，这使我们假设BLM需要 被拴系到Mcm 6上，以将BLM的ATP酶/解旋酶活性限制在Mcm 6附近。 复制体加上BLM的能力，以解开G-四链体（G4）和他们的存在，在整个 人类基因组，包括约90%的复制起点，我们提出BLM被Mcm 6招募， 展开DNA结构（i）在复制起点，以促进G1/S转换（目的1）和（ii）在整个 基因组在未受干扰的S期期间调节复制体进展（Aim 2）。我们分离出了一组 BLM突变体在G1期或S期或两者中特异性地不能与Mcm 6相互作用，以鉴定单独的 BLM/Mcm 6相互作用在G1和S期的功能并确定复制相关的有丝分裂 缺陷此外，我们将使用生物物理方法和分子动力学模拟来确定 通过BLM的G4解旋机制（目的3）。完成这些研究将描绘一个主要的新功能， BLM在未受干扰的DNA复制中，除了其在DNA双链断裂修复中的既定作用外， DNA损伤后复制叉重新启动，并确定其G4期解旋机制。我们的发现将 提供了一个重大进展，我们的机制，防止染色体不稳定性的理解， 不受干扰的细胞，提高我们对染色体断裂综合征和癌症的理解 易感性

PROJECT SUMMARY The RecQ-like DNA helicase BLM is known for its critical role in the response to and repair of DNA-double- strand breaks in mammalian cells. Disruption of BLM activity causes Bloom’s syndrome, which is characterized by extreme cancer risk, short stature, and an average life expectancy of 25 years. Cancer susceptibility, chromosome breakage and other cellular defects are currently explained by the lack of BLM’s activity in the DNA-damage response and homologous recombination. In this proposal we are testing the hypothesis that BLM plays critical roles in DNA replication initiation and elongation to maintain chromosome stability in unperturbed cells. This hypothesis is based on extensive preliminary data, including an unbiased screen of the mid-S-phase proteome that led to the discovery that chromatin-bound BLM directly interacts with the Mcm6 subunit of chromatin-bound Mcm2-7. Notably, two distinct binding sites in BLM and Mcm6 differentially regulate complex formation in G1 and S-phase, and disruption of the BLM/Mcm6 interaction in S-phase, but not in G1, leads to supra-normal DNA replication speed. Aberrant acceleration of DNA replication speed beyond a safe limit is emerging as a mechanism that causes DNA damage and kills certain types of cancer cells. Our preliminary findings suggest that the BLM/Mcm6 interaction acts as a novel, negative regulator of DNA replication in human cells. That cells lacking BLM do not exhibit increased replication speed suggests that acceleration of replication requires the BLM protein, leading us to hypothesize that BLM needs to be tethered to Mcm6 to restrict the ATPase/helicase activity of BLM to the immediate vicinity of the replisome. Together with BLM’s ability to unwind G-quadruplexes (G4s) and their presence throughout the human genome, including at ~90% of origins of replication, we propose that BLM is recruited by Mcm6 to unfold DNA structures (i) at replication origins to facilitate the G1/S transition (Aim 1) and (ii) throughout the genome to regulate replisome progression during unperturbed S-phase (Aim 2). We have isolated a set of BLM mutants that specifically fail to interact with Mcm6 in G1 or S-phase, or both, to identify the separate functions of the BLM/Mcm6 interaction in G1 and S-phase and to determine replication-associated mitotic defects. Further, we will use biophysical approaches and molecular dynamics simulations to determine the mechanism of G4 unwinding by BLM (Aim 3). Completing these studies will delineate a major new function for BLM in unperturbed DNA replication, besides its established role in DNA double-strand break repair and replication fork restart after DNA damage, and determine its mechanism of G4 unwinding. Our findings will provide a major advance in our understanding of the mechanisms that prevent chromosome instability in unperturbed cells and improve our understanding of chromosome breakage syndromes and cancer predisposition.