Spatial regulation of DNA damage tolerance protein Rad5 interconnects genome stability maintenance and proteostasis networks.

The Rad5/HLTF protein has a central role in the tolerance to DNA damage by mediating an error-free mode of bypassing unrepaired DNA lesions, and is therefore critical for the maintenance of genome stability. We show in this work that, following cellular stress, Rad5 is regulated by relocalization into two types of nuclear foci that coexist within the same cell, which we termed 'S' and 'I'. Rad5 S-foci form in response to genotoxic stress and are associated with Rad5's function in maintaining genome stability, whereas I-foci form in the presence of proteotoxic stress and are related to Rad5's own proteostasis. Rad5 accumulates into S-foci at DNA damage tolerance sites by liquid-liquid phase separation, while I-foci constitute sites of chaperone-mediated sequestration of Rad5 at the intranuclear quality control compartment (INQ). Relocalization of Rad5 into each type of foci involves different pathways and recruitment mechanisms, but in both cases is driven by the evolutionarily conserved E2 ubiquitin-conjugating enzyme Rad6. This coordinated differential relocalization of Rad5 interconnects DNA damage response and proteostasis networks, highlighting the importance of studying these homeostasis mechanisms in tandem. Spatial regulation of Rad5 under cellular stress conditions thus provides a useful biological model to study cellular homeostasis as a whole.

Fluorescence Microscopy for Analysis of Relocalization of Structure-Specific Endonucleases.

The analysis of protein relocalization by fluorescence microscopy has been important for studying processes involved in genome integrity maintenance at the cellular level. Structure-specific endonucleases are required for genome stability, and work in budding yeast has revealed that these proteins accumulate and colocalize at discrete subnuclear foci following DNA damage. Here we describe protocols for fluorescence microscopy analysis of live budding-yeast cells containing fluorescent-tagged proteins that have been useful for the study of endonuclease relocalization during the cell cycle and under DNA-damaging conditions, all of which can be extended to the analysis of other proteins.

Mus81-Mms4 endonuclease is an Esc2-STUbL-Cullin8 mitotic substrate impacting on genome integrity.

The Mus81-Mms4 nuclease is activated in G2/M via Mms4 phosphorylation to allow resolution of persistent recombination structures. However, the fate of the activated phosphorylated Mms4 remains unknown. Here we find that Mms4 is engaged by (poly)SUMOylation and ubiquitylation and targeted for proteasome degradation, a process linked to the previously described Mms4 phosphorylation cycle. Mms4 is a mitotic substrate for the SUMO-Targeted Ubiquitin ligase Slx5/8, the SUMO-like domain-containing protein Esc2, and the Mms1-Cul8 ubiquitin ligase. In the absence of these activities, phosphorylated Mms4 accumulates on chromatin in an active state in the next G1, subsequently causing abnormal processing of replication-associated recombination intermediates and delaying the activation of the DNA damage checkpoint. Mus81-Mms4 mutants that stabilize phosphorylated Mms4 have similar detrimental effects on genome integrity. Overall, our findings highlight a replication protection function for Esc2-STUbL-Cul8 and emphasize the importance for genome stability of resetting phosphorylated Mms4 from one cycle to another.

Prevention of unwanted recombination at damaged replication forks.

Homologous recombination is essential for the maintenance of genome integrity but must be strictly controlled to avoid dangerous outcomes that produce the opposite effect, genomic instability. During unperturbed chromosome replication, recombination is globally inhibited at ongoing DNA replication forks, which helps to prevent deleterious genomic rearrangements. This inhibition is carried out by Srs2, a helicase that binds to SUMOylated PCNA and has an anti-recombinogenic function at replication forks. However, at damaged stalled forks, Srs2 is counteracted and DNA lesion bypass can be achieved by recombination-mediated template switching. In budding yeast, template switching is dependent on Rad5. In the absence of this protein, replication forks stall in the presence of DNA lesions and cells die. Recently, we showed that in cells lacking Rad5 that are exposed to DNA damage or replicative stress, elimination of the conserved Mgs1/WRNIP1 ATPase allows an alternative mode of DNA damage bypass that is driven by recombination and facilitates completion of chromosome replication and cell viability. We have proposed that Mgs1 is important to prevent a potentially harmful salvage pathway of recombination at damaged stalled forks. In this review, we summarize our current understanding of how unwanted recombination is prevented at damaged stalled replication forks.

The Mgs1/WRNIP1 ATPase is required to prevent a recombination salvage pathway at damaged replication forks.

DNA damage tolerance (DDT) is crucial for genome integrity maintenance. DDT is mainly carried out by template switch recombination, an error-free mode of overcoming DNA lesions, or translesion DNA synthesis, which is error-prone. Here, we investigated the role of Mgs1/WRNIP1 in modulating DDT. Using budding yeast, we found that elimination of Mgs1 in cells lacking Rad5, an essential protein for DDT, activates an alternative mode of DNA damage bypass, driven by recombination, which allows chromosome replication and cell viability under stress conditions that block DNA replication forks. This salvage pathway is RAD52 and RAD59 dependent, requires the DNA polymerase δ and PCNA modification at K164, and is enabled by Esc2 and the PCNA unloader Elg1, being inhibited when Mgs1 is present. We propose that Mgs1 is necessary to prevent a potentially toxic recombination salvage pathway at sites of perturbed replication, which, in turn, favors Rad5-dependent template switching, thus helping to preserve genome stability.