Mechanism of NEIL3-dependent ICL repair

NEIL3依赖性ICL修复机制

• 批准号: 9757810
• 负责人: Daniel Semlow
• 金额: $ 9万
• 依托单位: HARVARD MEDICAL SCHOOL
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-09-01 至 2020-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9757810
• 关键词: Address; Advisory Committees; Affinity; Aging; Autoimmunity; Biochemical Genetics; Biochemistry; Bypass; Cell Culture Techniques; Cells; Cleaved cell; Coupled; DNA; DNA Damage; DNA Double Strand Break; DNA Repair; DNA biosynthesis; DNA glycosylase; DNA lesion; DNA replication fork; Defect; Development; Disease; Fanconi Anemia pathway; Fanconi' s Anemia; Genes; Genetic Diseases; Genome; Genomic Instability; Genomics; Glycosides; Goals; Head; Health; Human; Laboratories; Left; Lesion; Link; Maintenance; Malignant Neoplasms; Mass Spectrum Analysis; Mentors; Mentorship; Methods; Modeling; Molecular; Mutagens; Mutation; NEIL3 gene; Nucleotides; Pancytopenia; Pathway interactions; Phase; Physiological; Plasmids; Predisposition; Process; Proliferating; Psoralens; Refractory; Reporting; Resistance; Resource Development; S Phase; Site; Structure; Surgical incisions; Syndrome; System; Techniques; Testing; Therapeutic Intervention; Training; Tube; Vertebral column; Work; Xenopus; cancer cell; cancer genetics; career development; chemical genetics; chemotherapy; crosslink; egg; environmental agent; established cell line; gene function; homologous recombination; human disease; medical schools; novel; nuclease; nucleobase; outcome forecast; overexpression; reconstitution; recruit; repaired; replication stress; response; therapy design

Summary During each division, the cell must quickly and accurately replicate its genome. This process, however, is challenged by constant insults to DNA. DNA interstrand cross-links (ICLs) are genomic lesions that covalently link the two strands of DNA and block replication. If left unrepaired, these lesions can induce genomic instability, a hallmark of cancer. Although ICLs are generated by a variety of exogenous and endogenous agents, the structures of specific ICLs that arise spontaneously in cells are unknown. In proliferating cells, ICL repair occurs predominately in S phase. In the classic ICL repair pathway, repair requires replication fork convergence at an ICL and the cross-linked DNA strands are unhooked by nucleolytic incisions that generate a DNA double stranded break (DSB) intermediate. This DSB is then repaired by homologous recombination. Importantly, mutations in genes that function in this repair pathway cause the bone marrow failure and cancer predisposition syndrome Fanconi anemia (FA). Recently, an alternative ICL repair pathway that depends on the NEIL3 DNA glycosylase has been discovered. Like the FA pathway, the NEIL3 pathway requires replication fork convergence at an ICL. However, unlike the FA pathway, the NEIL3 pathway does not involve formation of a DSB intermediate. Instead, NEIL3 unhooks ICLs by cleaving one of the N-glycosyl bonds of the cross-linked nucleobases, generating an abasic site that can be bypassed by translesion synthesis. Unhooking by the NEIL3 pathway is therefore faster and less complicated than unhooking by the FA pathway and is the preferred ICL repair pathway for a subset of lesions. In this proposal, complementary biochemical, genetic, and analytical approaches will be used to investigate the mechanism of NEIL3-dependent ICL repair. Aim 1 seeks to determine how replication forks activate NEIL3-dependent unhooking using Xenopus egg extracts that recapitulate ICL repair. Aim 2 proposes to investigate how the NEIL3 and FA pathways are coordinated to allow efficient ICL repair in humans using a recently established cell line model. Finally, Aim 3 will address the question of which endogenous forms of DNA damage are targeted by ICL repair pathways through the development of a novel mass spectrometry approach to discover DNA lesions in cells. The mentored phase of this work will be undertaken at Harvard Medical School under the mentorship of Dr. Johannes Walter and an assembled advisory committee. The applicant will supplement previous training in biochemistry with additional training in cell culture and analytical mass spectrometry techniques with the goal of investigating the formation and repair of endogenous DNA lesions as the head of an independent laboratory. The applicant's goals will be facilitated by the rich experimental and career development resources of Harvard Medical School. Overall, this work has the potential to significantly impact human health. By understanding the mechanisms of ICL repair, it may be possible to design interventions that sensitize cancer cells to chemotherapy or mitigate the molecular defects that cause FA and other diseases.

总结 在每次分裂过程中，细胞必须快速准确地复制其基因组。然而，这个过程是 不断受到DNA损伤的挑战DNA链间交联（ICL）是基因组损伤， 连接两条DNA链并阻止复制。如果不修复，这些病变可以诱导基因组 不稳定是癌症的标志虽然ICL是由多种外源性和内源性的 由于这些药物的存在，细胞中自发产生的特定ICL的结构尚不清楚。在增殖细胞中，ICL 修复主要发生在S期。在经典的ICL修复途径中，修复需要复制叉 在ICL处聚合，并且交联的DNA链被溶核切口解开， DNA双链断裂（DSB）中间体。然后通过同源重组修复该DSB。 重要的是，在这一修复途径中起作用的基因突变会导致骨髓衰竭和癌症。 易感综合征范可尼贫血（FA）。最近，一种替代ICL修复途径， 发现了NEIL 3 DNA糖基化酶。与FA途径一样，NEIL 3途径需要 ICL上的复制分叉收敛。然而，与FA途径不同，NEIL 3途径不涉及 形成DSB中间体。相反，NEIL 3通过切割ICLs的一个N-糖基键来解开ICLs。 交联的核碱基，产生可以通过跨损伤合成绕过的脱碱基位点。脱钩 因此，通过NEIL 3途径的脱钩比通过FA途径的脱钩更快且更简单， 一个亚组病变的首选ICL修复途径。在这项建议中，补充生物化学，遗传， 分析方法将用于研究NEIL 3依赖性ICL修复的机制。目标1寻求 使用非洲爪蟾卵提取物来确定复制叉如何激活NEIL 3依赖性脱钩， 概括ICL修复。目的2提出研究NEIL 3和FA通路如何协调， 允许使用最近建立的细胞系模型在人类中进行有效的ICL修复。最后，目标3将解决 ICL修复途径通过以下途径靶向哪种内源性形式的DNA损伤的问题： 开发了一种新的质谱方法来发现细胞中的DNA损伤。指导阶段 这项工作将在哈佛医学院进行，由约翰内斯·沃尔特博士和一位 召集咨询委员会。申请人将补充以前的生物化学培训， 细胞培养和分析质谱技术的培训，目的是研究 和修复内源性DNA损伤作为一个独立的实验室的负责人。申请人的目标将是 借助哈佛医学院丰富的实验和职业发展资源。总体而言，这 工作有可能对人类健康产生重大影响。通过了解ICL修复的机制， 设计干预措施，使癌细胞对化疗敏感或减轻癌细胞的分子毒性， 导致FA和其他疾病的缺陷。