Mechanisms and Functions of Iron-Sulfur Helicases in DNA repair

项目摘要 DNA损伤是无法避免的，因为正常细胞环境中的元素，包括水， 氧气会造成损伤。因此，DNA修复途径对于维持基因组稳定性至关重要 以及预防与DNA损伤诱导的诱变相关的疾病。即使有功能性DNA修复 当遇到这些途径时，一些DNA损伤将不可避免地逃脱修复并阻止DNA复制。 复制体细胞有专门的途径来绕过或修复DNA损伤，阻止复制， 复制继续进行。DNA解旋酶是DNA修复所必需的酶之一，这些酶的作用是： 无论是在DNA复制过程中还是在DNA复制过程之外。含铁硫DNA解旋酶的Rad 3/XPD家族 在DNA修复和维持基因组稳定中起重要作用。有四个家庭成员在 人、XPD、FANCJ（a.k.a. BRIP 1）、DDX 11（也称为ChlR 1）和RTEL 1，这对人类至关重要。 健康，如与每个基因突变相关的疾病所证明的。遗传性疾病与 Rad 3/XPD家族解旋酶中的突变通常与基因组不稳定性相关， 癌症和许多其他疾病。该提案将定义生物化学和分子机制， 在大肠杆菌中新发现的Rad 3/XPD解旋酶家族成员YoaA， 修复DNA复制过程中的DNA损伤。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DNA复制的试剂在大肠杆菌中的应用大肠杆菌，他们确定了两个基因，yoaA和 holC，它们共同作用使细胞对AZT产生耐受性。蛋白质序列预测和我们的初步结果 确认第一个基因yoaA编码Fe-S解旋酶。第二个基因，holC，编码的c亚基的 DNA聚合酶Ⅲ全酶（pol Ⅲ HE）与E.大肠杆菌复制酶修复AZT损伤。然而，在这方面， 我们的初步结果揭示了c作为YoaA解旋酶亚基的一种新功能， c在修复AZT损伤的途径中与YoaA解旋酶而不是pol III HE一起起作用。主要 这个建议的前提是YoaA和c构成了一种DNA解旋酶，参与了修复 在停滞的复制叉处损坏的3'末端。我们的目标是：1）表征YoaA·c蛋白，2） 表征YoaA·c的解旋酶和底物偏好，以及3）定义YoaA·c与解旋酶之间的功能相互作用。 YoaA和c的体外研究，并开发一种关键试剂来研究这些体内功能相互作用。这项建议 将提供YoaA·c解旋酶的第一个生化表征，YoaA·c解旋酶是Rad 3/XPD家族的成员， 解旋酶通过维持基因组稳定性在人类健康中发挥关键作用。

PROJECT SUMMARY DNA damage cannot be prevented because elements in a normal cellular environment including water and oxygen contribute to damage. Therefore, DNA repair pathways are essential for maintaining genomic stability and preventing diseases associated with DNA damage-induced mutagenesis. Even with functional DNA repair pathways, some DNA damage will inevitably escape repair and block DNA replication when encountered by the replisome. Cells have specialized pathways to bypass or repair DNA damage that blocks replication to allow replication to proceed. DNA helicases are among the enzymes essential for DNA repair, and these enzymes act both during and outside of DNA replication. The Rad3/XPD family of iron-sulfur (Fe-S)-containing DNA helicases plays an important role in DNA repair and maintaining genome stability. There are four family members in humans, XPD, FANCJ (a.k.a. BRIP1), DDX11 (a.k.a. ChlR1), and RTEL1, that are crucially important for human health as evidenced by diseases associated with mutations in each of the genes. Genetic disorders linked to mutations in Rad3/XPD family helicases are typically associated with genome instability, a predisposition to cancer, and a number of other pathologies. This proposal will define biochemical and molecular mechanisms for a newly discovered member of the Rad3/XPD helicase family in Escherichia coli, YoaA, that plays a role in repairing DNA damage during DNA replication. Our collaborators in the Lovett laboratory use 3’-azido- 3’deoxythymidine (AZT) as a reagent to block DNA replication in E. coli, and they identified two genes, yoaA and holC, that work together to give cells tolerance to AZT. Protein sequence predicts, and our preliminary results confirm, that the first gene, yoaA, encodes an Fe-S helicase. The second gene, holC, encodes the c subunit of DNA polymerase III holoenzyme (pol III HE) implicating the E. coli replicase in repair of AZT lesions. However, our preliminary results have uncovered a novel function for c as a subunit of the YoaA helicase, and we propose that c functions with the YoaA helicase rather than pol III HE in a pathway that repairs AZT lesions. The main premise of this proposal 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 and substrate preferences for YoaA•c, and 3) define functional interactions between YoaA and c in vitro and develop a key reagent to investigate these functional interactions in vivo. This 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.