1 Identification of replisome-associated proteins in Drosophila embryos and cultured cells using iPOND coupled to quantitative mass spectrometry

Replication of the eukaryotic genome requires the assembly of thousands of replisomes that must work in concert to accurately replicate a cell’s genetic and epigenetic information. Defining replisome-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 replisome-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 416 replisome-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 the replisome to promote replication fork progression and maintain genome stability. Altogether, our work provides a valuable resource for those interested in the DNA replication, repair and chromatin assembly during development.