Abstract
Replication fork stalling and accumulation of single-stranded DNA trigger the S phase checkpoint, a signalling cascade that, in budding yeast, leads to the activation of the Rad53 kinase. Rad53 is essential in maintaining cell viability, but its targets of regulation are still partially unknown. Here we show that Rad53 drives the hyper-SUMOylation of Pol2, the catalytic subunit of DNA polymerase ε, principally following replication forks stalling induced by nucleotide depletion. Pol2 is the main target of SUMOylation within the replisome and its modification requires the SUMO-ligase Mms21, a subunit of the Smc5/6 complex. Moreover, the Smc5/6 complex co-purifies with Pol ε, independently of other replisome components. Finally, we map Pol2 SUMOylation to a single site within the N-terminal catalytic domain and identify a SUMO-interacting motif at the C-terminus of Pol2. These data suggest that the S phase checkpoint regulate Pol ε during replication stress through Pol2 SUMOylation and SUMO-binding ability
Highlights
The maintenance of genome stability requires the faithful and complete duplication of the chromosomes in each cell cycle
We show that the catalytic subunit of DNA Pol ε (Pol2), is the preferential target of modification with SUMO among the replication machinery components
This mono-SUMOylation depends on the S phase checkpoint factors Rad53, Mrc1 and Ctf18
Summary
The maintenance of genome stability requires the faithful and complete duplication of the chromosomes in each cell cycle. Pathways controlling the formation, activity and repair of replication forks play a key role in safeguarding cell viability and act as a bulwark against cell transformation [1, 2]. These pathways must be coordinated, integrating different stimuli within the cell, and the cross-talk between different post-translational modifications plays a critical role in their regulation. The initiation of chromosome replication is positively regulated by the phosphorylation of Mcm by DDK (Dbf4-dependent kinase) [3,4,5], and negatively controlled by SUMOylation of the origin-bound double hexamer of Mcm. The analysis of the cross-talk of different signalling pathways is often necessary to understand how complex processes are fine-tuned
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