DNA-Protein Cross-Links: Formation, Structural Identities, and Biological Outcomes.
DNA-Protein Cross-Links: Formation, Structural Identities, and Biological Outcomes.
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DNA蛋白交联：形成，结构身份和生物学结果。
DOI：
10.1021/acs.accounts.5b00056
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发表时间：
2015-06-16
影响因子：
18.3
通讯作者：
Ji S
中科院分区：
文献类型：
--
作者：
Tretyakova NY;Groehler A 4th;Ji S
非共价 DNA-蛋白质相互作用是正常细胞功能的核心。在真核细胞中，基因组 DNA 包裹在组蛋白八聚体周围，以便在细胞核中进行染色体包装。调节蛋白因子与 DNA 的结合可指导复制、控制转录并介导细胞对 DNA 损伤的反应。由于它们在所有涉及 DNA 的细胞过程中具有重要意义，动态 DNA-蛋白质相互作用是细胞生存所必需的，它们的破坏可能会产生严重的生物学后果。当细胞蛋白质在暴露于各种内源性、环境和化疗药物后被共价捕获在 DNA 链上时，就会形成 DNA-蛋白质交联 (DPC)。由于内源性暴露于活性氧和脂质过氧化产物以及正常的细胞代谢，DPC 逐渐在大脑和心脏组织中积累。在用化疗药物、过渡金属离子和代谢激活的致癌物治疗后，发现了一系列结构多样的 DPC。由于其相当大的尺寸和螺旋扭曲的性质，DPC 会干扰复制和转录机器的进展，从而阻碍遗传信息的忠实表达，可能导致突变和致癌。基于质谱的研究已经确定了数百种蛋白质，这些蛋白质在活性氧、致癌代谢物和抗肿瘤药物存在的情况下可以与核 DNA 交联。虽然这些蛋白质中的许多（包括组蛋白、转录因子和修复蛋白）是已知的 DNA 结合伴侣，但没有记录的 DNA 亲和力的其他基因产物也参与 DPC 形成。此外，DNA 内的多个位点可以作为交联的目标，包括鸟嘌呤的 N7、胸腺嘧啶的 C-5 甲基以及鸟嘌呤、胞嘧啶和腺嘌呤的环外氨基。这种结构的复杂性使得 DPC 病变的结构和生物学研究变得复杂。已经开发出两种通用策略来创建含有结构明确的、位点特异性 DPC 的 DNA 链。将 DNA 修饰蛋白捕获在其 DNA 底物上的酶促方法是位点特异性且高效的，但不允许系统研究 DPC 损伤结构对其生物学结果的影响。 DPC 形成的合成方法基于含有蛋白质反应性非天然 DNA 碱基的寡核苷酸链的固相合成。后一种方法允许更广泛的蛋白质底物与 DNA 缀合，并为 DNA 内的附着位点提供更大的灵活性。在这篇文章中，我们概述了细胞中 DPC 形成的化学过程，描述了我们最近通过质谱法鉴定交联蛋白的努力，并讨论了制备含有结构明确、位点特异性 DPC 损伤的 DNA 链的各种方法。使用模型 DPC 进行的聚合酶旁路实验表明，这些大体积病变的生物学结果强烈依赖于肽/蛋白质的大小和 DNA 内的确切交联位点。未来的研究需要阐明 DPC 修复机制及其在活细胞中的生物学结果。
Non-covalent DNA-protein interactions are at the heart of normal cell function. In eukaryotic cells, genomic DNA is wrapped around histone octamers to allow for chromosomal packaging in the nucleus. Binding of regulatory protein factors to DNA directs replication, controls transcription, and mediates cellular responses to DNA damage. Because of their fundamental significance in all cellular processes involving DNA, dynamic DNA-protein interactions are required for cell survival, and their disruption is likely to have serious biological consequences. DNA-protein cross-links (DPCs) form when cellular proteins become covalently trapped on DNA strands upon exposure to various endogenous, environmental and chemotherapeutic agents. DPCs progressively accumulate in the brain and heart tissues as a result of endogenous exposure to reactive oxygen species and lipid peroxidation products, as well as normal cellular metabolism. A range of structurally diverse DPCs are found following treatment with chemotherapeutic drugs, transition metal ions, and metabolically activated carcinogens. Because of their considerable size and their helix-distorting nature, DPCs interfere with the progression of replication and transcription machineries and hence hamper the faithful expression of genetic information, potentially contributing to mutagenesis and carcinogenesis. Mass spectrometry-based studies have identified hundreds of proteins that can become cross-linked to nuclear DNA in the presence of reactive oxygen species, carcinogen metabolites, and antitumor drugs. While many of these proteins including histones, transcription factors, and repair proteins are known DNA binding partners, other gene products with no documented affinity for DNA also participate in DPC formation. Furthermore, multiple sites within DNA can be targeted for cross-linking including the N7 of guanine, the C-5 methyl group of thymine, and the exocyclic amino groups of guanine, cytosine, and adenine. This structural complexity complicates structural and biological studies of DPC lesions. Two general strategies have been developed for creating DNA strands containing structurally defined, site-specific DPCs. Enzymatic methodologies that trap DNA modifying proteins on their DNA substrate are site specific and efficient, but do not allow for systematic studies of DPC lesion structure on their biological outcomes. Synthetic methodologies for DPC formation are based on solid phase synthesis of oligonucleotide strands containing protein-reactive unnatural DNA bases. The latter approach allows for a wider range of protein substrates to be conjugated to DNA and affords a greater flexibility for the attachment sites within DNA. In this Account, we outline the chemistry of DPC formation in cells, describe our recent efforts to identify the cross-linked proteins by mass spectrometry, and discuss various methodologies for preparing DNA strands containing structurally defined, site specific DPC lesions. Polymerase bypass experiments conducted with model DPCs indicate that the biological outcomes of these bulky lesions are strongly dependent on the peptide/protein size and the exact cross-linking site within DNA. Future studies are needed to elucidate the mechanisms of DPC repair and their biological outcomes in living cells.