Mechanism of NEIL3-dependent ICL repair

NEIL3依赖性ICL修复机制

• 批准号: 10207669
• 负责人: Daniel Semlow
• 金额: $ 24.9万
• 依托单位: CALIFORNIA INSTITUTE OF TECHNOLOGY
• 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 2023-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10207669
• 关键词: Address; Aging; Autoimmunity; Binding; Biochemical; Biological Assay; Bypass; C-terminal; Cells; Chemicals; Cleaved cell; Coupled; DNA; DNA Damage; DNA Double Strand Break; DNA Repair; DNA Repair Disorder; DNA Repair Pathway; DNA biosynthesis; DNA glycosylase; DNA lesion; DNA replication fork; Data; Defect; Development; Disease; Fanconi Anemia pathway; Fanconi' s Anemia; Genetic Diseases; Genome; Genomic Instability; Genomics; Glycosides; Human; Individual; Left; Lesion; Link; Malignant Neoplasms; Maps; Mass Spectrum Analysis; Mediating; Methodology; Molecular; Mutation; NEIL3 gene; Nucleotides; Pancytopenia; Pathway interactions; Physiological; Process; Prognosis; Proliferating; Recombinants; Refractory; Reporting; Resolution; Role; S Phase; Site; Structure; Surgical incisions; Syndrome; System; Testing; Therapeutic Intervention; Time; Tube; Vertebral column; Work; Xenopus; cancer cell; cancer predisposition; chemotherapy; chromatin immunoprecipitation; crosslink; disease disparity; egg; environmental agent; gene function; helicase; homologous recombination; human disease; insight; novel; nuclease; nucleobase; overexpression; reconstitution; recruit; repaired; replication stress; response; single molecule; stoichiometry; therapy design

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 particularly toxic genomic lesions that covalently link the two strands of DNA. If left unrepaired, these lesions can block replication and 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, we discovered an alternative ICL repair pathway that depends on the NEIL3 DNA glycosylase. Like the FA pathway, the NEIL3 pathway is activated by ubiquitylation of the replicative CMG helicase upon 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 crosslinked 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 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 the dynamics of NEIL3 at individual replication forks using single molecule approaches. 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. 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.

在每一次分裂过程中，细胞必须快速而准确地复制其基因组。这个过程， 然而，它受到了持续不断的DNA侮辱的挑战。DNA链间交链(ICL)是 尤其是将DNA的两条链共价连接的有毒基因组损伤。如果不修理，这些 病变可以阻止复制并诱导基因组不稳定，这是癌症的一个标志。尽管ICL 是由各种外源性和内源性因素产生的，特定ICL的结构 在细胞中自发产生的是未知的。在增殖细胞中，ICL修复主要发生在 在S阶段。在经典的ICL修复途径中，修复需要ICL处的复制分叉收敛 而交联的DNA链被核溶解的切口所解钩，从而产生DNA双链 搁浅断裂(DSB)中间体。然后通过同源重组修复这一DSB。 重要的是，在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，我们发现了一种替代方案 依赖于NEIL3 DNA糖基酶的ICL修复途径。与FA途径一样，NEIL3 途径是由复制的CMG解旋酶泛素化激活的分叉收敛在一个 ICL.然而，与FA途径不同，NEIL3途径不涉及DSB的形成 中级的。相反，NEIL3通过切割交联物的N-糖基键之一来解钩ICL 碱基，产生一个可被跨损伤合成绕过的基本位置。 因此，通过NEIL3途径解钩比通过 FA途径，是部分皮损的首选ICL修复途径。在这份提案中， 将使用互补的生化和分析方法来研究这种机制。 NEIL3依赖的ICL修复。目标1试图确定复制叉子如何激活NEIL3- 依赖解钩使用非洲爪哇卵萃取物，概括ICL修复。目标2建议 使用单分子方法研究NEIL3在单个复制分叉上的动态。 最后，目标3将解决针对哪些内源性形式的DNA损伤的问题 通过ICL修复途径开发的一种新的质谱学方法来发现 细胞的DNA损伤。通过了解ICL修复的机制，有可能设计出 使癌细胞对化疗敏感的干预措施或减轻分子缺陷 导致FA和其他疾病。