Mechanisms and Functions of Iron-Sulfur Helicase in DNA Repair

项目摘要 DNA复制经常被DNA损伤或其他障碍物（例如转录复合物）阻断， 才能让复制继续进行专门的途径对于拯救停滞的DNA至关重要 复制分叉以允许复制继续进行。我们在洛维特实验室的合作者使用3 '-叠氮基- 3 '脱氧胸苷（AZT）作为阻断大肠杆菌DNA复制的试剂，他们确定了两个 基因yoaA和holC共同作用，使细胞对AZT产生耐受性。蛋白质序列预测， 生化结果证实，第一个基因yoaA编码一种含铁硫（Fe-S）的DNA解旋酶， Rad 3/XPD家族第二个基因holC编码DNA聚合酶III全酶的c亚基 我们发现了c作为YoaA解旋酶亚基的一种新功能。DNA解旋酶，包括 Rad 3/XPD家族的含Fe-S的DNA解旋酶，在维持基因组稳定性中发挥重要作用， 参与DNA修复和挽救停滞的复制叉。Rad 3/XPD家族有四个成员， 人、XPD、FANCJ（a.k.a. BRIP 1）、DDX 11（也称为ChlR 1）和RTEL 1，这对于 人体健康与这些解旋酶突变相关的遗传性疾病通常与基因组 不稳定性、癌症易感性和许多其他病理。我们的提案定义了 新发现的E. coli家族成员YoaA.主要 前提是YoaA和c构成DNA解旋酶，其参与修复受损的3'末端， 已停止的复制分叉。我们的目标是：1）表征YoaA·c蛋白，2）表征解旋酶 YoaA·c的活性和底物偏好，以及3）确定c如何在体外促进YoaA活性， 并开发用于测定作为YoaA亚基的c相对于DNA的功能的cy融合蛋白 体内聚合酶III全酶。我们的母提案将提供第一个生物化学表征的 YoaA·c解旋酶是Rad 3/XPD解旋酶家族的一员，在人类健康中发挥着重要作用。 维持基因组的稳定性。本设备补充要求购买LUMICKS M型疏水阀 用宽场荧光光镊系统测量YoaA·c（和 YoaA）在单分子水平上。这一采购将直接支持《公约》目标2和3的实验， 我们的原始提案调查YoaA的机制和c对解旋酶的贡献， YoaA的活动。到目前为止，XPD是唯一一个使用这种方法表征的Rad 3/XPD家族成员。 一种单分子方法。确定这些家庭活动的异同 对于了解它们在正常条件下的功能和疾病中的功能障碍是重要的 states.此外，这种单分子方法是高度互补的合奏实验，我们 目前正在做的，并将提供一个更全面的看法YoaA-c解旋酶比任何一个 这一方法本身就能增强我们项目的影响力。

PROJECT SUMMARY DNA replication is frequently blocked by DNA damage or other obstacles (e.g. transcription complexes) that must be overcome to allow replication to continue. Specialized pathways are essential for rescuing stalled DNA replication forks to allow replication to proceed. Our collaborators in the Lovett laboratory use 3’-azido- 3’deoxythymidine (AZT) as a reagent to block DNA replication in Escherichia coli, and they identified two genes, yoaA and holC, that work together to give cells tolerance to AZT. Protein sequence predicts, and our biochemical results confirm, that the first gene, yoaA, encodes an iron-sulfur (Fe-S)-containing DNA helicase in the Rad3/XPD family. The second gene, holC, encodes the c subunit of the DNA polymerase III holoenzyme and we have uncovered a novel function for c as a subunit of the YoaA helicase. DNA helicases, including the Rad3/XPD family of Fe-S-containing DNA helicases, play essential roles in maintaining genome stability by participating in DNA repair and rescuing stalled replication forks. There are four Rad3/XPD family members in humans, XPD, FANCJ (a.k.a. BRIP1), DDX11 (a.k.a. ChlR1), and RTEL1, that are crucially important for human health. Genetic disorders linked to mutations in these helicases are typically associated with genome instability, a predisposition to cancer, and a number of other pathologies. Our parent proposal defines biochemical and molecular mechanisms for the newly discovered E. coli family member, YoaA. The main premise is that YoaA and c constitute a DNA helicase that is involved in the repair of damaged 3’ ends at stalled replication forks. Our aims are to: 1) characterize the YoaA•c protein, 2) characterize the helicase activities and substrate preferences for YoaA•c, and 3) determine how c contributes to YoaA activities in vitro, and to develop a cy fusion protein for assaying functions of c as a subunit of YoaA versus the DNA polymerase III holoenzyme in vivo. Our parent proposal will provide the first biochemical characterization of the YoaA•c helicase, a member of the Rad3/XPD family of helicases which play critical roles in human health by maintaining genome stability. This equipment supplement requests the purchase of a LUMICKS M-trap optical tweezer system with wide-field fluorescence to measure helicase activities of YoaA•c (and YoaA) at the single-molecule level. This purchase will directly support the experiments in Aims 2 and 3 of our parent proposal investigating the mechanism of YoaA and contributions that c makes to the helicase activities of YoaA. To date, XPD is the only Rad3/XPD family member that has been characterized using this type of single-molecule methodology. Identifying similarities and differences in the activities of these family members is important for understanding their functions under normal conditions and dysfunction in disease states. Moreover, this single-molecule approach is highly complementary to the ensemble experiments that we are currently doing and together will provide a more comprehensive view of the YoaA-c helicase than either approach alone would give, thus strengthening the impact of our project.