Mechanisms and Functions of Iron-Sulfur Helicase in DNA Repair

铁硫解旋酶在 DNA 修复中的机制和功能

• 批准号: 10581194
• 负责人: Linda B Bloom
• 金额: $ 20万
• 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/10581194
• 关键词: 2' -Deoxythymidine; Amino Acid Sequence; Biochemical; Biological Assay; Cells; Chimeric Proteins; Complex; DNA Damage; DNA Polymerase III; DNA Repair; DNA biosynthesis; DNA replication fork; Disease; Equipment; Escherichia coli; Family; Family member; Fluorescence; Functional disorder; Genes; Genetic Diseases; Genetic Transcription; Genome Stability; Genomic Instability; Health; Holoenzymes; Human; Iron; Laboratories; Link; Malignant Neoplasms; Measures; Methodology; Molecular; Mutation; Parents; Pathology; Pathway interactions; Play; Predisposition; Reagent; Role; Sulfur; System; Work; citrate carrier; experimental study; helicase; in vitro activity; in vivo; laser tweezer; member; novel; optical traps; preference; repaired; single molecule

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.

项目概要 DNA 复制经常受到 DNA 损伤或其他障碍（例如转录复合物）的阻碍， 必须克服这个问题才能继续复制。特殊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，他们鉴定了两种 yoaA 和 holC 基因共同作用，赋予细胞对 AZT 的耐受性。蛋白质序列预测，以及我们的 生化结果证实，第一个基因 yoaA 编码含铁硫 (Fe-S) 的 DNA 解旋酶 Rad3/XPD 系列。第二个基因 holC，编码 DNA 聚合酶 III 全酶的 c 亚基 我们还发现了 c 作为 YoaA 解旋酶亚基的新功能。 DNA 解旋酶，包括 Rad3/XPD 家族含有 Fe-S 的 DNA 解旋酶，通过以下方式在维持基因组稳定性方面发挥重要作用： 参与 DNA 修复并拯救停滞的复制叉。 Rad3/XPD 家族有四个成员 人类、XPD、FANCJ（又名 BRIP1）、DDX11（又名 ChlR1）和 RTEL1，对于 人类健康。与这些解旋酶突变相关的遗传性疾病通常与基因组相关 不稳定、易患癌症和许多其他病症。我们的父提案定义了 新发现的大肠杆菌家族成员 YoaA 的生化和分子机制。主要 前提是 YoaA 和 c 构成 DNA 解旋酶，参与修复受损的 3' 末端 复制叉停滞。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， 并开发一种 cy 融合蛋白，用于分析作为 YoaA 亚基的 c 与 DNA 的功能 体内聚合酶III全酶。我们的母提案将提供第一个生化特征 YoaA•c 解旋酶，Rad3/XPD 解旋酶家族的成员，通过以下方式在人类健康中发挥着关键作用： 维持基因组稳定性。本设备补充要求购买 LUMICKS M-trap 具有宽场荧光的光镊系统，用于测量 YoaA•c 的解旋酶活性（以及 YoaA）在单分子水平上。此次购买将直接支持目标 2 和 3 的实验 我们的母提案调查了 YoaA 的机制以及 c 对解旋酶的贡献 YoaA 的活动。迄今为止，XPD 是唯一使用此方法进行表征的 Rad3/XPD 家族成员 单分子方法的类型。识别这些家庭活动的异同 成员对于了解其在正常条件下的功能和疾病中的功能障碍非常重要 州。此外，这种单分子方法与我们的整体实验高度互补。 目前正在做的事情，将共同提供比任何一个都更全面的 YoaA-c 解旋酶视图 仅靠这种方法就能增强我们项目的影响力。