Mechanisms and Functions of Iron-Sulfur Helicases in DNA repair

项目总结 DNA损伤是无法防止的，因为正常细胞环境中的元素包括水和 氧气会造成损害。因此，DNA修复途径对于维持基因组稳定性是必不可少的 以及预防与DNA损伤诱导的突变相关的疾病。即使是有功能的DNA修复 一些DNA损伤将不可避免地逃脱修复并阻止DNA复制，当遇到 复制体。细胞有专门的途径绕过或修复阻止复制的DNA损伤，从而 以继续进行复制。Dna解旋酶是dna修复所必需的酶之一，这些酶起作用。 无论是在DNA复制期间还是在DNA复制之外。含铁硫(铁-S)解旋酶的RAD3/XPD家族 在DNA修复和维持基因组稳定性方面起着重要作用。这里有四个家庭成员 人类，XPD，FANCJ(又名BRIP1)、DDX11(又名Chlr1)和RTEL1，它们对人类至关重要 健康的证据是与每个基因突变相关的疾病。遗传性疾病与 RAD3/XPD家族解旋酶的突变通常与基因组不稳定有关，这是一种易患 癌症，以及其他一些病理疾病。这项提议将定义生物化学和分子机制 在大肠杆菌中新发现的RAD3/XPD解旋酶家族成员YoaA，它在 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- 3‘脱氧胸苷(AZT)作为阻断DNA在大肠杆菌中复制的试剂，他们发现了yoaA和yoaA两个基因。 它们共同作用，使细胞对AZT具有耐受性。蛋白质序列预测，以及我们的初步结果 确认第一个基因yoaA编码一个铁S解旋酶。第二个基因HOLC编码C亚基。 DNA聚合酶III全酶(PolIII HE)在AZT损伤修复中涉及大肠杆菌复制酶。然而， 我们的初步结果揭示了c作为YoaA解旋酶的一个亚单位的新功能，我们建议 在修复AZT损伤的途径中，c与YoaA解旋酶一起发挥作用，而不是PolIII HE。主 这一提议的前提是YoaA和c构成dna解旋酶，该dna解旋酶参与修复 停滞复制叉处的3‘端已损坏。我们的目标是：1)鉴定YoaA·c蛋白，2) 表征YoaA·c的解旋酶和底物偏好，以及3)确定 并开发了一种关键试剂来研究体内这些功能的相互作用。这项建议 将首次提供YoaA·c解旋酶的生化特征，YoaA.c解旋酶是RAD3/XPD家族的成员 解旋酶通过维持基因组的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.