Li Zhiming, Hua Xu, Serra-Cardona Albert, Xu Xiaowei, Zhang Zhiguo
Institute for Cancer Genetics, Columbia University Irving Medical Center, New York, NY, USA.
Herbert Irving Comprehensive Cancer Center, Columbia University Irving Medical Center, New York, NY, USA.
Nat Protoc. 2021 May;16(5):2698-2721. doi: 10.1038/s41596-021-00520-6. Epub 2021 Apr 28.
Faithful duplication of both genetic and epigenetic information is essential for all eukaryotic cells. DNA replication initiates from replication origins and proceeds bidirectionally but asymmetrically, with the leading strand being synthesized continuously and the lagging strand discontinuously as Okazaki fragments by distinct DNA polymerases. Unraveling the underlying mechanisms of chromatin replication at both strands is crucial to better understand DNA replication and its coupled processes, including nucleosome assembly, sister chromatid cohesion and DNA mismatch repair. Here we describe the enrichment and sequencing of protein-associated nascent DNA (eSPAN) method to analyze the enrichment of proteins of interest, including histones and their modifications at replicating chromatin in a strand-specific manner in mammalian cells. Briefly, cells are pulsed with the thymidine analog bromodeoxyuridine to label newly synthesized DNA. After cell permeabilization, the target proteins are sequentially bound by antibodies and protein A-fused transposase, which digests and tags genomic DNA of interest once activated by magnesium. The strand specificity is preserved through oligo-replacement. Finally, the resulting double-strand DNA is denatured and immunoprecipitated with antibodies against bromodeoxyuridine to enrich nascent DNA associated with proteins of interest. After PCR amplification and next-generation sequencing, the mapped reads are used to calculate the relative enrichment of the target proteins around replication origins. Compared with alternative methods, the eSPAN protocol is simple, cost-effective and sensitive, even in a relatively small number of mammalian cells. The whole procedures from cell collection to generation of sequencing-ready libraries can be completed in 2 days.
忠实复制遗传信息和表观遗传信息对所有真核细胞来说都至关重要。DNA复制从复制起点开始，双向但不对称地进行，前导链由不同的DNA聚合酶连续合成，滞后链则以冈崎片段的形式间断合成。阐明两条链上染色质复制的潜在机制对于更好地理解DNA复制及其相关过程（包括核小体组装、姐妹染色单体黏连和DNA错配修复）至关重要。在此，我们描述了蛋白质相关新生DNA富集与测序（eSPAN）方法，以在哺乳动物细胞中以链特异性方式分析感兴趣的蛋白质（包括组蛋白及其在复制染色质上的修饰）的富集情况。简而言之，用胸苷类似物溴脱氧尿苷对细胞进行脉冲处理，以标记新合成的DNA。细胞通透后，目标蛋白依次与抗体和蛋白A融合转座酶结合，蛋白A融合转座酶一旦被镁激活，就会消化并标记感兴趣的基因组DNA。通过寡核苷酸置换保留链特异性。最后，将得到的双链DNA变性，并用抗溴脱氧尿苷抗体进行免疫沉淀，以富集与感兴趣的蛋白质相关的新生DNA。经过PCR扩增和二代测序后，映射的读数用于计算复制起点周围目标蛋白的相对富集情况。与其他方法相比，eSPAN方案简单、经济高效且灵敏，即使在相对少量的哺乳动物细胞中也是如此。从细胞收集到生成测序就绪文库的整个过程可在2天内完成。
