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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, Author summary Chromosome duplication is essential for cell proliferation. Mistakes during this process introduce mutations, duplications, deletions and rearrangements of the genetic information. Cells have evolved a monitoring pathway that supervises DNA replication, called S phase checkpoint; this regulates many aspects of the cell biology in response of defects during DNA replication, so to promote the complete and faithful completion of DNA replication once the obstacles are eliminated. While many details of the S phase checkpoint are known, how this pathway regulates the machine copying the DNA is still largely poorly understood. Here we present a novel mechanism of action of the S phase checkpoint preferentially observed in response to replication forks stalling. 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. Moreover, Pol ε is bound and SUMOylated by the Smc5/6 complex. Finally, we identified the site of the Pol2 modification and a SUMO binding-motif, suggesting a possible mechanism of action for this modification.

Mosquitoes are responsible for the transmission of many clinically important arboviruses that cause significant levels of annual mortality and socioeconomic health burden worldwide. Deciphering the mechanisms by which mosquitoes modulate arbovirus infection is crucial to understand how viral-host interactions promote vector transmission and human disease. SUMOylation is a post-translational modification that leads to the covalent attachment of the Small Ubiquitin-like MOdifier (SUMO) protein to host factors, which in turn can modulate their stability, interaction networks, sub-cellular localisation, and biochemical function. While the SUMOylation pathway is known to play a key role in the regulation of host immune defences to virus infection in humans, the importance of this pathway during arbovirus infection in mosquito vectors, such as Aedes aegypti (Ae. aegypti), remains unknown. Here we characterise the sequence, structure, biochemical properties, and tissue-specific expression profiles of component proteins of the Ae. aegypti SUMOylation pathway. We demonstrate significant biochemical differences between Ae. aegypti and Homo sapiens SUMOylation pathways and identify cell-type specific patterns of SUMO expression in Ae. aegypti tissues known to support arbovirus replication. Importantly, depletion of core SUMOylation effector proteins (SUMO, Ubc9 and PIAS) in Ae. aegypti cells led to enhanced levels of arbovirus replication from three different families; Zika (Flaviviridae), Semliki Forest (Togaviridae), and Bunyamwera (Bunyaviridae) viruses. Our findings identify an important role for mosquito SUMOylation in the cellular restriction of arboviruses that may directly influence vector competence and transmission of clinically important arboviruses., Author summary Half the world’s population is at risk of infection from arboviruses transmitted by mosquitoes. Deciphering the viral-host interactions that influence the outcome of arbovirus infection in mosquitoes is beneficial to the development of future vector control strategies to limit arbovirus transmission and viral emergence within the human population. Similar to humans, mosquitoes possess different immune pathways to limit the replication of arboviruses. While the Small Ubiquitin-like MOdifier (SUMO) pathway is known to play an important role in the regulation of immune defences to viral infection in humans, the influence of this pathway during arbovirus infection in mosquito cells is currently unknown. Here we define the conservation, biochemical activity, and tissue distribution of the core effector proteins of the Aedes aegypti SUMOylation pathway. We show that the mosquito SUMOylation pathway plays a broadly antiviral role against a wide range of clinically important arboviruses, including Zika, Semliki Forest, and Bunyamwera viruses. Our findings identify SUMOylation as an important component of the antiviral response to arbovirus infection in mosquito cells.