DNA Damage Tolerance Pathway Choice Through Uls1 Modulation of Srs2 SUMOylation in Saccharomyces cerevisiae

Abstract

DNA damage tolerance and homologous recombination pathways function to bypass replication-blocking lesions and ensure completion of DNA replication. However, inappropriate activation of these pathways may lead to increased mutagenesis or formation of deleterious recombination intermediates, often leading to cell death or cancer formation in higher organisms. Post-translational modifications of PCNA regulate the choice of repair pathways at replication forks. Its monoubiquitination favors translesion synthesis, while polyubiquitination stimulates template switching. Srs2 helicase binds to small ubiquitin-related modifier (SUMO)-modified PCNA to suppress a subset of Rad51-dependent homologous recombination. Conversely, SUMOylation of Srs2 attenuates its interaction with PCNA Sgs1 helicase and Mus81 endonuclease are crucial for disentanglement of repair intermediates at the replication fork. Deletion of both genes is lethal and can be rescued by inactivation of Rad51-dependent homologous recombination. Here we show that Uls1, a member of the Swi2/Snf2 family of ATPases and a SUMO-targeted ubiquitin ligase, physically interacts with both PCNA and Srs2, and promotes Srs2 binding to PCNA by downregulating Srs2-SUMO levels at replication forks. We also identify deletion of as a suppressor of ΔΔ synthetic lethality and hypothesize that Δ mutation results in a partial inactivation of the homologous recombination pathway, detrimental in cells devoid of both Sgs1 and Mus81 We thus propose that Uls1 contributes to the pathway where intermediates generated at replication forks are dismantled by Srs2 bound to SUMO-PCNA. Upon deletion, accumulating Srs2-SUMO-unable to bind PCNA-takes part in an alternative PCNA-independent recombination repair salvage pathway(s).

SUMO is a pervasive regulator of meiosis

“…Notably, Ubc4/5 work with SUMO-targeted ubiquitin ligases (STUbLs) that target poly-SUMOylated substrates ( Sriramachandran and Dohmen, 2014 ; Uzunova et al, 2007 ). SUMO also modified five ubiquitin E3 ligases, Ubr1, Ufo1, Uls1, Gid7, and Ufd4, involved in a variety processes including the N-end rule, catabolite-degradation of fructose-1,6-bisphosphatase, transcription, cell cycle, and genome maintenance ( Bao et al, 2015 ; Baranes-Bachar et al, 2008 ; Finley et al, 2012 ; Kramarz et al, 2017 ; Lin et al, 2015 ; Sriramachandran and Dohmen, 2014 ; Varshavsky, 1997 ). Notable is Uls1, a putative STUbL that is thought to compete with a second STUbL, Slx5-Slx8, to displace from DNA poly-SUMOylated proteins that have been rendered defective or inactive ( Sriramachandran and Dohmen, 2014 ; Tan et al, 2013 ; Wei et al, 2017 ).…”

“…Notable is Uls1, a putative STUbL that is thought to compete with a second STUbL, Slx5-Slx8, to displace from DNA poly-SUMOylated proteins that have been rendered defective or inactive ( Sriramachandran and Dohmen, 2014 ; Tan et al, 2013 ; Wei et al, 2017 ). Inferred Uls1 targets, all of which were SUMOylated in meiosis, include Top2 ( Wei et al, 2017 ), H2A.Z ( Takahashi et al, 2017 ), Rap1 ( Lescasse et al, 2013 ), Srs2 ( Kramarz et al, 2017 ), Rad51 ( Chi et al, 2011 ) and possibly Dmc1 ( Dresser et al, 1997 ; Figure 4A ). The ubiquitin-dependent segregase, Cdc48/p97, can target proteins co-modified by ubiquitin and SUMO ( Bergink et al, 2013 ; Nie et al, 2012 ).…”

Intricate SUMO-based control of the homologous recombination machinery

“…This may provide a way to curb Srs2-mediated inhibition of Rad51 in situations when HR is needed to rescue replication defects. Adding to this picture, Uls1, which also interacts with Slx5/8, was recently shown to associate with Srs2 and restrain its levels as well (Kramarz et al 2017). It will be interesting to gain a deeper understanding of the interplay between Uls1, Slx5/8, and Esc2 in Srs2 control, such as whether they act sequentially or at distinct DNA damage sites.…”

Access to PCNA by Srs2 and Elg1 Controls the Choice between Alternative Repair Pathways in Saccharomyces cerevisiae

“…In this study, we have used a minimalist approach and left out additional levels of regulation, such as Srs2 modifications and cellular regulators. Additional players that have an impact on pathway choice are Uls1 ( 54 ), Esc2 ( 29 ), Mph1/Mhf1-2 ( 55 , 56 ), and others. Future work will center on the role played by these proteins, as well as the choice between TS and TLS, both of which require PCNA ubiquitylation.…”