Model Genetic Systems to Study DNA Repair

建立遗传系统模型来研究 DNA 修复

• 批准号: 8335918
• 负责人: Robert Brosh
• 金额: $ 22.86万
• 依托单位: NATIONAL INSTITUTE ON AGING
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/8335918
• 关键词: Alkylating Agents; Basic Science; Biological; Biological Models; Cell Line; Cell Proliferation; Cell physiology; Cells; Chickens; Chromosomal Stability; Critical Pathways; DNA; DNA Damage; DNA Repair; DNA Repair Pathway; DNA Structure; DNA biosynthesis; DNA repair protein; Diagnosis; Disease; Embryo; Evolution; Fanconi' s Anemia; Fibroblasts; G-Quartets; Genetic; Genetic Models; Genome; Genome Stability; Genomic Instability; Genotype; Helicase Gene; Hereditary Disease; Homeostasis; Human; Human Cell Line; Human Genetics; Individual; Knockout Mice; Link; Maintenance; Malignant Neoplasms; Molecular; Mus; Mutation; Normal Cell; Orthologous Gene; Pathway interactions; Patients; Pharmaceutical Preparations; Phenotype; Premature aging syndrome; RNA Interference; Radiation; Resistance; Role; Stress; Structure; System; Werner Syndrome; Work; Yeasts; base; cancer cell; cancer therapy; chemotherapy; clinically relevant; helicase; in vivo; insight; mutant; novel; repaired; small molecule; tumor

Using an approach known as RNA interference, we have specifically depleted human cells of the FANCJ DNA helicase and characterized the sensitivity of the cells to a small molecule compound that stabilizes G-quadruplex DNA structures. This work enabled us to elucidate a novel function of the FANCJ helicase in the manintenace of chromosomal stability. Since individuals carrying homozygous mutations in the FANCJ helicase gene have a genetic disorder known as Fanconi Anemia characterized by genomic instability and cancer, we believe our results shed new insights to the cellular pathways of FANCJ that serve to counter replciational stress due to alterante DNA structures such as G-quadruplexes that arise in vivo. We have used isogenic pairs of mutant and corrected chicken DT40 cells as well as human cells for genetic complementation studies. The mutant cell lines were used for structure-function studies of patient derived helicase-inactivating mutations. This work enabled us to define genotype-phenotype relationships between clinically relevant mutations and human genetic diseases. Another aspect of this work was to used genetically defined cancer and normal cell lines and small molecules to probed the molecualr and cellular functions of the Werner syndrome helicase. These efforts enabled us to determine that inhibition of WRN helicase activity inhibits cell proliferation and its DNA repair function in vivo. These efforts in the basic sciences help to advance the idea that DNA repair proteins may be targeted in anti-cancer therapy to increase the sensitivity of tumors to DNA damaging chemotherapy drugs or radiation. We have also employed yeast as a model genetic system to study the role of the Werner syndrome helicase in DNA replciation and repair. This work has enabled us to characterize the catalytic requirements of WRN in a defined genetic DNA repair pathway that operates to provide cellular resistance to alkylating agents which impose replicational stress. Lastly, we have utilized mouse as a model genetic system to characterize the role of a helicase ortholog (RECQ1) found in humans whose biological significance is not well understood. Characterization of th eprimary mouse embryonic fibroblasts from RECQ1 knockout mice has revealed that the RECQ1 helicase has unique and important roles in genomic stability maintenance.

使用一种称为RNA干扰的方法，我们特别耗尽了FANCJ DNA解旋酶的人类细胞，并表征了细胞对稳定G- Qu-Quadruplex DNA结构的小分子化合物的敏感性。 这项工作使我们能够阐明染色体稳定性的FANCJ解旋酶的新功能。 由于在FANCJ解放酶基因中携带纯合突变的个体具有一种以基因组不稳定性和癌症为特征的遗传疾病，因此我们相信我们的结果为FANCH的细胞途径提供了新的见解，该途径是由诸如g-Quarplexees诸如Arise arise vivo的anterante dna结构所引起的替代DNA结构所引起的。 我们已经使用了突变体和校正的鸡DT40细胞以及人类细胞的等生成对进行遗传互补研究。 突变细胞系用于患者衍生的解旋酶灭活突变的结构功能研究。 这项工作使我们能够定义临床相关突变与人类遗传疾病之间的基因型 - 表型关系。 这项工作的另一个方面是使用遗传定义的癌症，正常细胞系和小分子来探测Werner综合征解旋酶的分子和细胞功能。 这些努力使我们能够确定抑制WRN解旋酶活性会抑制细胞增殖及其在体内的DNA修复功能。 基本科学中的这些努力有助于提高抗癌治疗中DNA修复蛋白的目标，以提高肿瘤对DNA对DNA损害化学疗法药物或放射线的敏感性。 我们还利用酵母作为模型遗传系统来研究Werner综合征解旋酶在DNA反式围和修复中的作用。 这项工作使我们能够表征WRN在定义的遗传DNA修复途径中的催化需求，该途径可为烷基化剂提供施加复制应激的烷基化剂的细胞耐药性。 最后，我们利用小鼠作为模型遗传系统来表征在人类中发现的解旋酶直系同源物（RECQ1）的作用，该人的生物学意义尚不清楚。 RECQ1基因敲除小鼠的电子胚胎胚胎成纤维细胞的表征表明，RECQ1解旋酶在基因组稳定性维持中具有独特而重要的作用。