Improved detection of DNA replication fork-associated proteins via iPOND2-DRIPPER

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 di-glycine 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.

INSTRUMENT(S):

ORGANISM(S): Mus Musculus (mouse)

TISSUE(S): Cell Culture

SUBMITTER: Ya-Chu Chang  

LAB HEAD: Anja Katrin Bielinsky

PROVIDER: PXD050381 | Pride | 2025-05-06

REPOSITORIES: Pride