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.

概括 在每个分裂过程中，细胞必须快速准确地复制其基因组。但是，这个过程是 不断侮辱DNA的挑战。 DNA链间交联（ICL）是共价性的基因组病变 链接DNA的两个链和块复制。如果未修复，这些病变会诱导基因组 不稳定，癌症的标志。尽管ICL是由多种外源和内生产生的 代理，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维修途径中，维修需要复制叉 ICL的收敛性和交联的DNA链被产生A的核解切口解开 DNA双链断裂（DSB）中间体。然后通过同源重组来修复此DSB。 重要的是，在此修复途径中起作用的基因突变会导致骨髓衰竭和癌症 倾向综合征芬科尼贫血（FA）。最近，取决于替代的ICL修复途径 已经发现了Neil3 DNA糖基化酶。像FA通路一样，Neil3途径需要 ICL处的复制叉收敛。但是，与FA通路不同，Neil3途径不涉及 DSB中间体的形成。取而代 交联的核仁酶，产生一个可以通过跨性别合成绕过的无碱性位点。解开 因此 首选的ICL修复途径，用于一部分病变。在此提案中，互补的生化，遗传和 分析方法将用于研究Neil3依赖性ICL修复的机制。目标1寻求 确定复制叉如何使用Xenopus鸡蛋提取物激活neil3依赖性解钩 概括ICL修复。 AIM 2建议研究Neil3和FA途径如何协调到 使用最近建立的细胞系模型允许人类中有效的ICL修复。最后，AIM 3将解决 通过ICL修复途径针对哪种内源性DNA损伤的问题 开发一种新型的质谱方法，以发现细胞中的DNA病变。指导的阶段 这项工作将在约翰内斯·沃尔特博士的指导下在哈佛医学院进行 组装咨询委员会。申请人将补充先前的生物化学培训 细胞培养和分析质谱技术的培训，目的是研究形成 并修复内源性DNA病变作为独立实验室的头部。申请人的目标将是 哈佛医学院丰富的实验和职业发展资源促进。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维修的机制 可能会设计干预措施，使癌细胞对化学疗法敏感或减轻分子 引起FA和其他疾病的缺陷。