Improved detection of DNA replication fork-associated proteins

Rebecca S Rivard; Ya-Chu Chang; Ryan L Ragland; Yee-Mon Thu; Muzaffer Kassab; Rahul Shubhra Mandal; Susan K Van Riper; Katarzyna Kulej; LeeAnn Higgins; Todd M Markowski; David Shang; Jack Hedberg; Luke Erber; Benjamin Garcia; Yue Chen; Anja-Katrin Bielinsky; Eric J Brown

doi:10.1016/j.celrep.2024.114178

Improved detection of DNA replication fork-associated proteins

Cell Rep. 2024 May 2;43(5):114178. doi: 10.1016/j.celrep.2024.114178. Online ahead of print.

Authors

Rebecca S Rivard¹, Ya-Chu Chang², Ryan L Ragland¹, Yee-Mon Thu², Muzaffer Kassab¹, Rahul Shubhra Mandal¹, Susan K Van Riper³, Katarzyna Kulej⁴, LeeAnn Higgins², Todd M Markowski², David Shang¹, Jack Hedberg², Luke Erber², Benjamin Garcia⁴, Yue Chen², Anja-Katrin Bielinsky⁵, Eric J Brown⁶

Affiliations

¹ Department of Cancer Biology and the Abramson Family Cancer Research Institute, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA.
² Department of Biochemistry, Molecular Biology and Biophysics, University of Minnesota, Minneapolis, MN, USA.
³ University of Minnesota Informatics Institute, University of Minnesota, Minneapolis, MN, USA.
⁴ Department of Biochemistry and Biophysics, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA.
⁵ Department of Biochemistry, Molecular Biology and Biophysics, University of Minnesota, Minneapolis, MN, USA. Electronic address: azu3jn@virginia.edu.
⁶ Department of Cancer Biology and the Abramson Family Cancer Research Institute, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA. Electronic address: brownej@upenn.edu.

PMID: 38703364
DOI: 10.1016/j.celrep.2024.114178

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.

Keywords: CP: Molecular biology; DNA repair; DNA replication; DRIPPER; iPOND; nuclear pore complex; p97; replication stress; replisome; ubiquination.