Department of Biological Sciences, Vanderbilt University, Nashville, TN, 37212, USA.
Department of Chemistry, Vanderbilt University, Nashville, TN, USA.
Sci Rep. 2022 Apr 28;12(1):6903. doi: 10.1038/s41598-022-10821-9.
Replication of the eukaryotic genome requires the formation of thousands of replication forks that must work in concert to accurately replicate the genetic and epigenetic information. Defining replication fork-associated proteins is a key step in understanding how genomes are replicated and repaired in the context of chromatin to maintain genome stability. To identify replication fork-associated proteins, we performed iPOND (Isolation of Proteins on Nascent DNA) coupled to quantitative mass spectrometry in Drosophila embryos and cultured cells. We identified 76 and 278 fork-associated proteins in post-MZT embryos and Drosophila cultured S2 cells, respectively. By performing a targeted screen of a subset of these proteins, we demonstrate that BRWD3, a targeting specificity factor for the DDB1/Cul4 ubiquitin ligase complex (CRL4), functions at or in close proximity to replication forks to promote fork progression and maintain genome stability. Altogether, our work provides a valuable resource for those interested in DNA replication, repair and chromatin assembly during development.
真核生物基因组的复制需要形成数千个复制叉，这些复制叉必须协同工作，以准确复制遗传和表观遗传信息。鉴定与复制叉相关的蛋白质是理解在染色质背景下基因组如何复制和修复以维持基因组稳定性的关键步骤。为了鉴定与复制叉相关的蛋白质，我们在果蝇胚胎和培养细胞中进行了 iPOND（新生 DNA 上的蛋白质分离）与定量质谱联用实验。我们分别在 MZT 后胚胎和果蝇培养的 S2 细胞中鉴定到了 76 种和 278 种与复制叉相关的蛋白质。通过对这些蛋白质中的一部分进行有针对性的筛选，我们证明了 BRWD3，一种 DDB1/Cul4 泛素连接酶复合物（CRL4）的靶向特异性因子，在复制叉处或附近发挥作用，以促进叉的推进并维持基因组稳定性。总的来说，我们的工作为那些对发育过程中 DNA 复制、修复和染色质组装感兴趣的人提供了有价值的资源。
