Slx8 Removes Pli1-Dependent Protein-SUMO Conjugates Including SUMOylated Topoisomerase I to Promote Genome Stability

Abstract

The SUMO-dependent ubiquitin ligase Slx8 plays key roles in promoting genome stability, including the processing of trapped Topoisomerase I (Top1) cleavage complexes and removal of toxic SUMO conjugates. We show that it is the latter function that constitutes Slx8's primary role in fission yeast. The SUMO conjugates in question are formed by the SUMO ligase Pli1, which is necessary for limiting spontaneous homologous recombination when Top1 is present. Surprisingly there is no requirement for Pli1 to limit recombination in the vicinity of a replication fork blocked at the programmed barrier RTS1. Notably, once committed to Pli1-mediated SUMOylation Slx8 becomes essential for genotoxin resistance, limiting both spontaneous and RTS1 induced recombination, and promoting normal chromosome segregation. We show that Slx8 removes Pli1-dependent Top1-SUMO conjugates and in doing so helps to constrain recombination at RTS1. Overall our data highlight how SUMOylation and SUMO-dependent ubiquitylation by the Pli1-Slx8 axis contribute in different ways to maintain genome stability.

Different proteomic strategies to identify genuine Small Ubiquitin-like MOdifier targets and their modification sites inTrypanosoma bruceiprocyclic forms

“…A high proportion of the proteins identified as SUMO targets belong to functionally related nuclear processes, such as DNA replication and repair, transcription, rRNA biogenesis, RNA processing and degradation, and chromatin remodelling, among others, suggesting that SUMO signalling could be mediated by several proteins within protein groups. Some well‐known SUMO targets in yeast and mammals have been identified in this study, like PCNA (Ulrich, ) and Topoisomerase IB (Mao et al ., ; Steinacher et al ., ) with four SUMOylation sites identified at the non‐conserved hydrophilic N‐terminal end of the large subunit (homologous to the core of the general monomeric enzyme). In kinetoplastids, Topoisomerase IB is a heterodimer and both subunits are essential for cell viability (Bakshi and Shapiro, ).…”

Schizosaccharomyces pombe Assays to Study Mitotic Recombination Outcomes

“…The Whitby lab has subsequently been influential in designing several variations of the tandem repeat assay and have been able to explain some of the more complex outcomes. These assays have been used to identify and characterize genes involved in DNA damage repair [67][68][69][70][71][72][73][74][75][76][77][78]. The investigators initially designed a system similar to the Schuchert and Kholi assay except that instead of ura4 + they placed a his3 + marker between the ade6 alleles ( Figure 3C) [79].…”

“…The investigators used this assay to study the function of various recombination genes in fork restart and to show that rescue of collapsed replication forks can cause BIR dependent template switching that can generate chromosomal rearrangements. [76,87,88,90,91]. A protocol was published with extensive details on the use of these elegant assays [92].…”

Slx5/Slx8 Promotes Replication Stress Tolerance by Facilitating Mitotic Progression

“…To date, only a few in vivo substrates of Slx5/8 and RNF4 have been experimentally determined. Those include mediator of DNA damage checkpoint 1 (MDC1), centromere protein-I (CENP-I), Fanconi anemia complementation group D2 and I (FACND2/FACNI) and Jumonji/ARID1 B (JARID1B) in mammalian cells (Galanty et al, 2012; Gibbs-Seymour et al, 2015; Hendriks et al, 2015; Luo et al, 2012; Mukhopadhyay et al, 2010; Yin et al, 2012), and modifier of transcription 1 (Mot1), MATα2 repressor, topoisomerase 1 (Top1), Siz1 and mitotic chromosome determinant 1 (Mcd1) in yeast (D’Ambrosio and Lavoie, 2014; Steinacher et al, 2013; Wang and Prelich, 2009; Westerbeck et al, 2014; Xie et al, 2010). …”