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Improved detection of DNA replication fork-associated proteins.
- Source :
- Cell Reports; May2024, Vol. 43 Issue 5, pN.PAG-N.PAG, 1p
- Publication Year :
- 2024
-
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. [Display omitted] • iPOND2-DRIPPER improves dynamic range for detection of replisome proteins • iPOND2-DRIPPER identifies sites of ubiquitination without di-Gly enrichment • iPOND2-DRIPPER expands upon previously identified replisome components Rivard et al. report that a combination of density-based replisome enrichment and label-free quantitative mass spectrometry (iPOND2-DRIPPER) substantially increases both replication and repair factor yields and the dynamic range of protein quantification. Using this method, the authors identify notable ubiquitination and spatiotemporal changes in replication-fork-associated proteins following replication stress. [ABSTRACT FROM AUTHOR]
Details
- Language :
- English
- ISSN :
- 26391856
- Volume :
- 43
- Issue :
- 5
- Database :
- Complementary Index
- Journal :
- Cell Reports
- Publication Type :
- Academic Journal
- Accession number :
- 177421029
- Full Text :
- https://doi.org/10.1016/j.celrep.2024.114178