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.

项目总结 DNA复制经常被DNA损伤或其他障碍(如转录复合体)阻止 必须克服这些问题，才能继续复制。特殊的途径对于拯救停滞的DNA是必不可少的 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- 3‘脱氧胸腺嘧啶核苷(AZT)作为阻断DNA复制的试剂在大肠杆菌中，他们鉴定了两个 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，我们的 生化结果证实，第一个基因yoaA编码一个含硫铁(Fe-S)的dna解旋酶。 RAD3/XPD家族。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解旋酶的一个亚单位的新功能。DNA解旋酶，包括 RAD3/XPD家族的含铁S的解旋酶，通过以下途径在维持基因组稳定中发挥重要作用 参与DNA修复和拯救停滞不前的复制叉子。有四个Rad3/XPD家庭成员在 人类，XPD，FANCJ(又名BRIP1)、DDX11(又名ChlR1)和RTEL1，它们对 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有关。 不稳定，易患癌症，以及许多其他病理因素。我们的母公司提案定义了 新发现的大肠杆菌家族成员YoaA的生化和分子机制。主 前提是YoaA和c组成DNA解旋酶，参与修复受损的3‘端 复制分叉停滞。我们的目标是：1)鉴定YoaA·c蛋白；2)鉴定解旋酶 YoaA·c的活性和底物偏好，以及3)c在体外如何影响YoaA的活性， 并开发了一种Cy融合蛋白，用于检测c作为YoaA亚单位与DNA的功能。 体内的聚合酶III全酶。我们的母公司提案将提供第一个生物化学特征 YoaA.c解旋酶是Rad3/XPD解旋酶家族的成员之一，它通过以下途径在人类健康中发挥关键作用 保持基因组的稳定。本设备补充申请购买LUMICKS M-TRAP 测定YoaA·c解旋酶活性的广场荧光光钳系统(和 YoaA)在单分子水平上。此次购买将直接支持的目标2和3的实验 我们的母公司建议研究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特性来表征Rad3/XPD家族成员的成员 单分子方法学的类型。找出这些家庭活动的异同 成员对于了解它们在正常情况下的功能和疾病中的功能障碍很重要 各州。此外，这种单分子方法是对我们的整体实验的高度补充 目前正在做的工作，并将提供一个比任何一个更全面的YoaA-c解旋酶的观点 单靠方法就能起到作用，从而加强我们项目的影响。