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

• Published in: Scientific reports Vol. 12; no. 1; pp. 6903 - 11
• Main Authors: Munden, Alexander, Wright, Madison T., Han, Dongsheng, Tirgar, Reyhaneh, Plate, Lars, Nordman, Jared T.
• Format: Journal Article
• Language: English
• Published: London Nature Publishing Group UK 28.04.2022; Nature Publishing Group; Nature Portfolio
• Subjects: 631/136; 631/45; 631/45/147; 631/80/103; Animals; Cells, Cultured; Chromatin - genetics; Chromatin remodeling; Deoxyribonucleic acid; DNA; DNA biosynthesis; DNA repair; DNA Replication; Drosophila; Drosophila - genetics; Embryos; Epigenetics; Genomes; Genomic Instability; Humanities and Social Sciences; Insects; Mass spectrometry; Mass Spectrometry - methods; Mass spectroscopy; multidisciplinary; Proteins; Replication forks; Science; Science (multidisciplinary); Scientific imaging; Ubiquitin; Ubiquitin-protein ligase
• ISSN: 2045-2322; 2045-2322
• 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.