Mechanism of NEIL3-dependent ICL repair

在每次分裂过程中，细胞必须快速准确地复制其基因组。这个过程， 然而，它受到了DNA不断损伤的挑战。DNA链间交联（ICL） 特别是共价连接两条DNA链的毒性基因组损伤。如果不进行修复， 病变可阻断复制并诱导基因组不稳定性，这是癌症的标志。虽然ICL 是由各种外源性和内源性因素产生的，特定ICL的结构 是未知的。在增殖细胞中，ICL修复主要发生在 处于S期。在经典的ICL修复途径中，修复需要ICL处的复制叉会聚 而交联的DNA链被溶核切口解开， 链断裂（DSB）中间体。然后通过同源重组修复该DSB。 重要的是，在这个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法 ICL修复途径依赖于NEIL 3 DNA糖基化酶。与FA途径一样，NEIL 3 途径是通过复制CMG解旋酶的泛素化激活的， ICL。然而，与FA途径不同，NEIL 3途径不涉及DSB的形成 中间体相反，NEIL 3通过切割交联的N-glycosyl键中的一个来解开ICL。 核碱基，产生可以通过跨损伤合成绕过的脱碱基位点。 因此，通过NEIL 3途径脱钩比通过NEIL 3途径脱钩更快且更不复杂。 FA途径，是一部分病变的首选ICL修复途径。在这一提议中， 补充的生物化学和分析方法将被用来调查的机制 NEIL 3依赖的ICL修复。目标1试图确定复制叉如何激活NEIL 3- 依赖脱钩使用爪蟾卵提取物，重演ICL修复。目标2建议， 使用单分子方法研究NEIL 3在单个复制叉处的动力学。 最后，目标3将解决的问题，其中内源性形式的DNA损伤的目标 通过ICL修复途径，通过开发一种新的质谱方法来发现 细胞中的DNA损伤。通过了解ICL修复的机制， 使癌细胞对化疗敏感或减轻 引起FA和其他疾病。

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.