Improved detection of DNA replication fork-associated proteins
Rebecca S. Rivard, Ya‐Chu Chang, Ryan L. Ragland, Yee Mon Thu, Muzaffer Ahmad Kassab, Rahul Shubhra Mandal, Susan K. Van Riper, Katarzyna Kulej, LeeAnn Higgins, Todd M. Markowski, David Shang, Jack Hedberg, Luke Erber, Benjamin A. García, Yue Chen, Anja‐Katrin Bielinsky, Eric J. Brown
Abstract
Innovative methods to retrieve proteins associated with actively replicating DNA have provided a glimpse into the molecular dynamics of replication fork stalling. We report that a combination of density-based replisome enrichment by isolating proteins on nascent DNA (iPOND2) and label-free quantitative mass spectrometry (iPOND2-DRIPPER) substantially increases both replication factor yields and the dynamic range of protein quantification. Replication protein abundance in retrieved nascent DNA is elevated up to 300-fold over post-replicative controls, and recruitment of replication stress factors upon fork stalling is observed at similar levels. The increased sensitivity of iPOND2-DRIPPER permits direct measurement of ubiquitination events without intervening retrieval of diglycine tryptic fragments of ubiquitin. Using this approach, we find that stalled replisomes stimulate the recruitment of a diverse cohort of DNA repair factors, including those associated with poly-K63-ubiquitination. Finally, we uncover the temporally controlled association of stalled replisomes with nuclear pore complex components and nuclear cytoskeleton networks.