Mechanisms and Functions of Iron-Sulfur Helicases in DNA repair

铁硫解旋酶在 DNA 修复中的机制和功能

• 批准号: 10493087
• 负责人: Linda B Bloom
• 金额: $ 29.57万
• 依托单位: UNIVERSITY OF FLORIDA
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-09-23 至 2025-08-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10493087
• 关键词: 2' -Deoxythymidine; ATP Hydrolysis; Amino Acid Sequence; Binding Sites; Biochemical; Biological Assay; Biological Models; Bypass; Cell physiology; Cells; Cellular Assay; Chimeric Proteins; Complex; DNA; DNA Binding; DNA Damage; DNA Polymerase III; DNA Repair; DNA Repair Enzymes; DNA Repair Pathway; DNA biosynthesis; DNA replication fork; DNA-Directed DNA Polymerase; Disease; Dissociation; Elements; Environment; Enzymes; Escherichia coli; Family; Family member; Genes; Genetic; Genetic Diseases; Genome Stability; Genomic DNA; Genomic Instability; Goals; Health; Hereditary Disease; Holoenzymes; Human; In Vitro; Iron; Kinetics; Laboratories; Lesion; Link; Malignant Neoplasms; Measures; Modeling; Molecular; Mutagenesis; Mutation; Oxygen; Pathology; Pathway interactions; Play; Predisposition; Proteins; Reagent; Role; Single-Stranded DNA; Site; Structure; Sulfur; Testing; Water; Work; Y protein; citrate carrier; ds-DNA; experimental study; helicase; in vivo; member; novel; polypeptide; preference; prevent; protein complex; protein protein interaction; repaired; replicase; stoichiometry

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.

项目概要 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至关重要 以及预防与 DNA 损伤诱导突变相关的疾病。即使有功能性 DNA 修复 途径中，一些 DNA 损伤在遇到 DNA 损伤时将不可避免地无法修复并阻止 DNA 复制。 复制体。细胞有专门的途径来绕过或修复 DNA 损伤，从而阻止复制，从而允许 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 DNA复制期间和之外。含铁硫 (Fe-S) 的 DNA 解旋酶 Rad3/XPD 家族 在DNA修复和维持基因组稳定性中发挥着重要作用。家里有四位家庭成员 人类、XPD、FANCJ（又名 BRIP1）、DDX11（又名 ChlR1）和 RTEL1，这些对人类至关重要 与每个基因突变相关的疾病证明了健康。与以下因素相关的遗传性疾病 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 癌症和许多其他病症。该提案将定义生物化学和分子机制 大肠杆菌中新发现的 Rad3/XPD 解旋酶家族成员 YoaA，在 修复DNA复制过程中的DNA损伤。我们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，他们鉴定了两个基因：yoaA 和 holC，共同作用使细胞对 AZT 产生耐受。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 Fe-S 解旋酶。第二个基因 holC，编码 c 亚基 DNA 聚合酶 III 全酶 (pol III HE) 涉及大肠杆菌复制酶修复 AZT 损伤。然而， 我们的初步结果发现了 c 作为 YoaA 解旋酶亚基的新功能，我们提出 c 在修复 AZT 损伤的途径中与 YoaA 解旋酶而不是 pol III HE 一起发挥作用。主要 该提议的前提是 YoaA 和 c 构成 DNA 解旋酶，参与修复 停滞的复制叉处的 3' 末端受损。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) 定义之间的功能相互作用 YoaA 和 c 在体外进行实验，并开发了一种关键试剂来研究这些体内功能相互作用。这个提议 将提供 YoaA•c 解旋酶的第一个生化特征，该解旋酶是 Rad3/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。