Stalled replication fork repair in cancer predisposition and cancertherapy

项目总结 在复制分叉停滞位置启动的无错误DNA修复是预防基因组的关键 细胞循环中的不稳定性。叉子修复失速的缺陷直接与癌症易感性有关。 以及其他人类疾病。与叉子修复失败相关的临床负担可能包括遗传性 乳腺癌和卵巢癌(HBOC)的易感性，鉴于BRCA1和BRCA2参与修复 复制叉停滞和范可尼贫血(FA)--一种罕见的常染色体隐性遗传(或X连锁)疾病 通过使几个FA基因中的任何一个失活。我们之前的工作确定了BRCA1和BRCA2的角色 在双链断裂(DSB)和停滞的叉子修复中调节HR。我们开发了创新的工具 用于量化哺乳动物复制停滞时的同源重组(HR)和其他修复结果 分叉，以及最近出现的损坏的复制分叉。这项提案的一个主要目标是定义基本的 失速叉子的修复机制。我们开发了一系列尖端工具来支持这项研究， 包括独特的、经验丰富的人力资源记者，他们可以区分无错误的短篇人力资源和错误- 倾向于“长道”HR--一种复制反应，类似于酵母中断裂诱导的复制。一件不同寻常的事 我们在停滞的叉子上观察到的异常复制反应，特别是在BRCA1突变细胞中是 -lt；10kb非同源串联复制(TD)的形成。在一个改变范式的发现中，我们发现 这些高度特异的结构变异形式也在人类BRCA1连锁的乳房中大量存在 和卵巢癌基因组。这项提案的一个主要目标是定义基因调控和充分 BRCA1突变细胞中失速分叉形成TD的机制。这个项目的成功将体现在 前所未有的细节调控哺乳动物失速(或折断)的叉子修复和它们的 与癌症易感性的关系。为了支持这一点，我们将开发分析DNA的新技术 失速哺乳动物的结构中间体、染色质对叉子失速的反应和蛋白质组成 复制分叉。这些分析研究还可能确定新的分子靶点，用于治疗乳腺癌和 卵巢癌。事实上，我们最近对BRCA1连锁TD形成机制的研究使我们 发现BRCA1和FANCM功能丧失突变之间的合成致命性相互作用。FANCM是一种 运动蛋白，因此，是一种ATPase。我们发现，单独消融FANCM ATPase活性(剩下的 细胞内完整和稳定的蛋白质)足以使BRCA1突变细胞致死。因此， FANCM可能是BRCA1相关癌症治疗的“可用药”靶点。在这里提出的工作中，我们将 定义这一发现的治疗潜力。在资助期内，我们希望作出重要的 在这一领域的发现，并为HBOC的新疗法打开大门，也许还有其他形式的癌症。

PROJECT SUMMARY Error-free DNA repair initiated at the sites of replication fork stalling is critical for the prevention of genomic instability in cycling cells. Defects in stalled fork repair have been directly implicated in cancer predisposition and other human diseases. The clinical burden associated with failed stalled fork repair may include hereditary breast and ovarian cancer (HBOC) predisposition, in light of the involvement of BRCA1 and BRCA2 in repair of stalled replication forks, and Fanconi Anemia (FA)—a rare, autosomal recessive (or X-linked) disease caused by inactivation of any one of several FA genes. Our work previously established roles for BRCA1 and BRCA2 in regulating HR at both double strand breaks (DSBs) and in stalled fork repair. We developed innovative tools for quantifying homologous recombination (HR) and other repair outcomes at stalled mammalian replication forks and, more recently, at broken replication forks. A major goal of this proposal is to define the fundamental mechanisms of repair of stalled forks. We have developed an array of cutting-edge tools to support this study, including unique, sophisticated HR reporters that can distinguish between error-free “short tract” HR and error- prone “long tract” HR—a replicative response analogous to break-induced replication in yeast. One unusual aberrant replicative response that we observe at stalled forks specifically in BRCA1 mutant cells is the formation of <10 kb non-homologous tandem duplications (TDs). In a paradigm-shifting discovery, we found that these highly specific forms of structural variation are also abundant in the human BRCA1-linked breast and ovarian cancer genome. A major goal of this proposal is to define the genetic regulation and full mechanism of TD formation at stalled forks in BRCA1 mutant cells. Success in this project will reveal in unprecedented detail the mechanisms that regulate mammalian stalled (or broken) fork repair and their relationship to cancer predisposition. In support of this, we will develop new techniques for analyzing DNA structural intermediates, chromatin responses to fork stalling and protein composition of the stalled mammalian replication fork. These analytical studies may also identify new molecular targets for therapy of breast and ovarian cancer. Indeed, our recent work on the mechanisms underlying formation of BRCA1-linked TDs led us to discover a synthetic lethal interaction between BRCA1 and FANCM loss-of-function mutations. FANCM is a motor protein and, hence, an ATPase. We find that ablation of FANCM ATPase activity alone (leaving the rest of the protein intact and stable within the cell) is sufficient to confer lethality on BRCA1 mutant cells. Thus, FANCM may be a “druggable” target for therapy in BRCA1-linked cancer. In work proposed herein, we will define the therapeutic potential of this discovery. During the funding period, we expect to make important discoveries in this field and to open the door to new therapies in HBOC and perhaps other forms of cancer.