Stalled replication fork repair in cancer predisposition and cancertherapy

项目摘要 在复制叉停滞位点启动的无错误DNA修复对于预防基因组损伤至关重要。 循环电池的不稳定性。停滞的分叉修复缺陷直接与癌症易感性有关 和其他人类疾病。临床负担与失败的失速叉修复可能包括遗传性 乳腺癌和卵巢癌（HBOC）易感性，根据BRCA 1和BRCA 2参与修复 范可尼贫血（FA）-一种罕见的常染色体隐性（或X连锁）疾病， 通过几个FA基因中的任何一个的失活。我们之前的工作确定了BRCA 1和BRCA 2的作用 在双链断裂（DSB）和分叉修复停滞时调节HR。我们开发了创新的工具 用于定量哺乳动物复制停滞时的同源重组（HR）和其他修复结果 forks，以及最近出现的损坏的复制forks。该提案的一个主要目标是确定 故障叉的修复机制。我们开发了一系列尖端工具来支持这项研究， 包括独特的，复杂的人力资源报告，可以区分无错误的“短道”人力资源和错误， 倾向于“长道”HR--一种类似于酵母中断裂诱导复制的复制反应。一个不寻常 我们在BRCA 1突变细胞中观察到的停滞分叉处的异常复制反应是 形成<10 kb的非同源串联重复（TD）。在一项改变范式的发现中，我们发现 这些高度特异性的结构变异形式在人类BRCA 1相关乳腺癌中也很丰富， 和卵巢癌基因组。该提案的一个主要目标是定义基因调控和完整的 BRCA 1突变细胞中停滞分叉处TD形成的机制。该项目的成功将在 前所未有的详细机制，调节哺乳动物停滞（或断裂）叉修复和他们的 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， 结构中间体，对叉停滞的染色质反应和停滞哺乳动物的蛋白质组成 复制分叉。这些分析研究还可以确定用于治疗乳腺癌和乳腺癌的新的分子靶点。 卵巢癌事实上，我们最近对BRCA 1相关TD形成机制的研究使我们 发现BRCA 1和FANCM功能丧失突变之间的合成致死相互作用。FANCM是一个 马达蛋白，因此，ATP酶。我们发现，FANCM ATP酶活性的单独消融（留下其余的）， 蛋白质在细胞内是完整和稳定的）足以对BRCA 1突变细胞产生致死性。因此，在本发明中， FANCM可能是BRCA 1相关癌症治疗的一个“药物”靶点。在本文提出的工作中，我们将 定义这一发现的治疗潜力。在融资期间，我们希望 这一领域的新发现，并为HBOC和其他形式的癌症的新疗法打开了大门。

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.