DNA replication is the process of producing two same replicas from one original DNA molecule. Small Ubiquitin-like Modifiers (SUMOs) are known to reduce replication stress, only a small number of relevant SUMO target proteins are known.
The SUMO E1 is a dimer consisting of SAE1 and SAE2. A single E2 enzyme, Ubc9, mediates conjugation of SUMO to all target proteins. SUMO E3 enzymes include PIAS protein family members and the nucleoporin RanBP2. Mice deficient for Ubc9 die at the early post-implantation stage showing DNA hypo-condensation and chromosomal aberrancies. Here we report identify a large scale of SUMOylated proteins.
We first used Ni-NTA purification to enrich His10-SUMO-2 conjugates from U2OS cells stably expressing His10-SUMO-2.
Following statistical analysis on five biological replicates, a total of 566 SUMO-2 targets were identified. We also uncovered sets of SUMO target proteins specifically up-regulated or down-regulated in response to replication stress.
We verified SUMOylation dynamics upon DNA replication stress by immunoblotting analysis for a subset of the identified dynamic SUMO-2 targets
To summary, this results uncover a comprehensive network of SUMO target proteins dealing with replication damage and provide a framework for detailed understanding of the role of SUMOylation to counteract replication stress.