基于生物化学交联技术的碱基切除DNA修复通路的机制与功能研究

DNA is challenged by both endogenous and exogenous agents, often resulting in cytotoxic and/or mutagenic adducts. Once damaged, celluar DNA must be promptly repaired. Base-excision repair (BER) is one major pathway during the cellular DNA repair process, and has attracted wide attention from scientists of many different fields. Within the BER pathway, the NEIL family DNA glycosylases have been shown to play important roles in the processes of neurogenesis, brain development, neoplasia and etc. It is widely believed that intermolecular interactions between NEIL family glycosylases and other components from the BER pathways are critical to maintain the proper functions of the NEIL glycosylases; thus, detailed knowledge about these transient interactions are crucial to our understanding of the BER pathway. However, many of these interactions are weak and fleeting, and protein/DNA and protein/protein complexes formed during the DNA repair process are too unstable to allow characterizations and investigations at the atomic level. Here we propose to develop and optimize a series of novel biochemical approaches and techniques, which can stabilize a lot of these weak interactions. Subsequently, both protein/DNA complexes and protein/protein complexes can be structurally characterized and detailed mechanisms of the NEIL family glycosylases can be investigated. In addition, the proposed techniques, which are based primarily on disulphide cross-linking, shoud be applicable to stabilize weak and transient interactions from other systems as well.

DNA时刻受到细胞内外因素的挑战,所产生的DNA损伤如不能被及时修复,将导致细胞死亡和癌症的发生。碱基切除修复是细胞在DNA修复过程中的一种重要机制,受到了广泛的关注。在这一生物通路中,NEIL家族的糖基化酶被发现在神经的形成、大脑的正常发育、避免肿瘤增生等方面都起着重要作用。NEIL家族的糖基化酶通过与通路中其他成员的复杂相互作用来实现对DNA损伤的及时修复；因此，要深入了解这一DNA修复机制，就必须对碱基切除修复通路中的众多瞬时相互作用进行研究。然而，许多这样的作用都是弱相互作用，所形成的蛋白质/DNA复合物、蛋白质复合物均不稳定，很难在原子水平上进行表征和研究。本项目将发展和运用一系列新颖的生物化学方法,稳定碱基切除修复通路中许多重要却瞬时的弱相互作用，通过复合物结构的鉴定对NEIL家族糖基化酶的分子机制进行研究。同时，本项目拟发展的二硫键交联技术也适用于稳定其他体系中的弱相互作用。

DNA 时刻受到细胞内外因素的挑战，所产生的 DNA 损伤如不能被及时修复,将导致细胞死亡和癌症的发生。碱基切除修复是细胞在DNA 修复过程中的一种重要机制，而其中NEIL家族的糖基化酶被发现在大脑的正常发育、避免肿瘤增生等方面都起着重要作用。NEIL家族的糖基化酶NEIL1识别并修复多种类型的DNA损伤，然而NEIL1蛋白质结合DNA的复合物结合力弱、稳定性差，因此NEIL1-DNA的复合物结构一直没有报道。在本项目中，我们通过解析人类NEIL1蛋白及含有不同类型底物双链DNA的复合物晶体结构，我们发现了NEIL1可以通过一个重要的柔性区域识别损伤的碱基。通过分子模拟，我们发现了NEIL1可以催化底物发生异构化，这个异构化对于NEIL1识别及催化底物的切除都是非常重要的。这种基于底物异构化的识别与催化机制是首次在酶催化反应中被发现。因此，该研究不但从原子水平上揭示了NEIL1特异性识别不同底物的机制，也提出了一种碱基切除修复的新机制。