Improved detection of DNA replication fork-associated proteins

Cell Rep. 2024 May 28;43(5):114178. doi: 10.1016/j.celrep.2024.114178. Epub 2024 May 2.

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.

MeSH terms

  • DNA / metabolism
  • DNA Repair
  • DNA Replication*
  • Humans
  • Ubiquitination*

Substances

  • DNA