Institute for Cancer Genetics, Columbia University, New York, NY 10032, USA.
Department of Biomedical Engineering, Columbia University, New York, NY 10027, USA.
Nucleic Acids Res. 2021 May 21;49(9):5074-5083. doi: 10.1093/nar/gkab271.
Silver nanoclusters (AgNCs) have outstanding physicochemical characteristics, including the ability to interact with proteins and DNA. Given the growing number of diagnostic and therapeutic applications of AgNCs, we evaluated the impact of AgNCs on DNA replication and DNA damage response in cell-free extracts prepared from unfertilized Xenopus laevis eggs. We find that, among a number of silver nanomaterials, AgNCs uniquely inhibited genomic DNA replication and abrogated the DNA replication checkpoint in cell-free extracts. AgNCs did not affect nuclear membrane or nucleosome assembly. AgNCs-supplemented extracts showed a strong defect in the loading of the mini chromosome maintenance (MCM) protein complex, the helicase that unwinds DNA ahead of replication forks. FLAG-AgNCs immunoprecipitation and mass spectrometry analysis of AgNCs associated proteins demonstrated direct interaction between MCM and AgNCs. Our studies indicate that AgNCs directly prevent the loading of MCM, blocking pre-replication complex (pre-RC) assembly and subsequent DNA replication initiation. Collectively, our findings broaden the scope of silver nanomaterials experimental applications, establishing AgNCs as a novel tool to study chromosomal DNA replication.
银纳米簇（AgNCs）具有出色的物理化学特性，包括与蛋白质和 DNA 相互作用的能力。鉴于 AgNCs 在诊断和治疗方面的应用越来越多，我们评估了 AgNCs 对未受精卵母细胞无细胞提取物中 DNA 复制和 DNA 损伤反应的影响。我们发现，在许多种银纳米材料中，AgNCs 独特地抑制了基因组 DNA 复制，并在无细胞提取物中破坏了 DNA 复制检查点。AgNCs 不影响核膜或核小体组装。AgNCs 补充的提取物在微染色体维持（MCM）蛋白复合物的加载中表现出严重缺陷，该复合物是复制叉前解开 DNA 的解旋酶。FLAG-AgNCs 免疫沉淀和 AgNCs 相关蛋白的质谱分析表明 MCM 和 AgNCs 之间存在直接相互作用。我们的研究表明，AgNCs 直接阻止 MCM 的加载，从而阻断预复制复合物（pre-RC）的组装和随后的 DNA 复制起始。总的来说，我们的发现拓宽了银纳米材料实验应用的范围，确立了 AgNCs 作为研究染色体 DNA 复制的新工具。
