A Flp-SUMO hybrid recombinase reveals multi-layered copy number control of a selfish DNA element through post-translational modification

Abstract

Mechanisms for highly efficient chromosome-associated equal segregation, and for maintenance of steady state copy number, are at the heart of the evolutionary success of the 2-micron plasmid as a stable multi-copy extra-chromosomal selfish DNA element present in the yeast nucleus. The Flp site-specific recombination system housed by the plasmid, which is central to plasmid copy number maintenance, is regulated at multiple levels. Transcription of the FLP gene is fine-tuned by the repressor function of the plasmid-coded partitioning proteins Rep1 and Rep2 and their antagonist Raf1, which is also plasmid-coded. In addition, the Flp protein is regulated by the host’s post-translational modification machinery. Utilizing a Flp-SUMO fusion protein, which functionally mimics naturally sumoylated Flp, we demonstrate that the modification signals ubiquitination of Flp, followed by its proteasome-mediated degradation. Furthermore, reduced binding affinity and cooperativity of the modified Flp decrease its association with the plasmid FRT (Flp recombination target) sites, and/or increase its dissociation from them. The resulting attenuation of strand cleavage and recombination events safeguards against runaway increase in plasmid copy number, which is deleterious to the host—and indirectly—to the plasmid. These results have broader relevance to potential mechanisms by which selfish genomes minimize fitness conflicts with host genomes by holding in check the extra genetic load they pose.

Irc20 Regulates the Yeast Endogenous 2-μm Plasmid Levels by Controlling Flp1

“…The increase in Flp1-induced nicks concomitant with a lack of regulatory control over HR, would lead to the aberrant processing of the replicated plasmids into monomers in daughter cells, thus HMW aggregate formation. This is consistent with that slx5 and slx8 still produces toxic 2-µm plasmid forms even when Flp-SUMO is expressed, despite being attenuated in strand cleavage (Ma et al, 2019). The reason would be deficient regulation on HR proteins.…”

“…The reduced turnover of Flp1 when irc20 is mutated is similar to that observed in siz1 siz2 , slx5 and slx8 which affect the SUMOylation and/or the subsequent ubiquitin dependent degradation of Flp1 (Ma et al, 2019). The resulting increase in copy number and formation of HMW aggregates is, however, different in case of irc20 mutants.…”

A natural variant of the essential host gene MMS21 restricts the parasitic 2-micron plasmid in Saccharomyces cerevisiae

“…The identification of MMS21 led us to initially suspect that this locus might represent another connection between the SUMO-ligation machinery and 2μ plasmid stability ( Dobson et al, 2005 ; Zhao et al, 2004 ; Pinder et al, 2013 ; Ma et al, 2019 ). However, the location of the Thr69Ile missense change at the binding interface between Mms21 and Smc5 ( Figure 5—figure supplement 3 ) suggested a mechanism that relies on the Smc5/6 complex rather than the catalytic RING domain.…”

“…2plasmid stability is frequently compromised in heterospecific (other species) hosts, suggesting it is actively adapting to maintain stability within its native host species 37 . Our discovery of a natural host variant of S. The identification of MMS21 led us to initially suspect that this locus might represent another connection between the SUMO-ligation machinery and 2plasmid stability 34,35,55,92 . However, the location of the Thr69Ile missense change at the binding interface between Mms21 and Smc5 (Figure supplement 3) suggested a mechanism that relies on the Smc5/6 complex rather than the catalytic RING domain.…”

A natural variant of the essential host geneMMS21restricts the parasitic 2-micron plasmid inSaccharomyces cerevisiae

“…Most studies of 2µ plasmid plasmids (including this one) have focused on the A-type variant that is most commonly found in laboratory strains. However, new sequencing studies have revealed that S. cerevisiae strains harbor a diverse set of 2µ plasmid The identification of MMS21 led us to initially suspect that this locus might represent another connection between the SUMO-ligation machinery and 2-micron plasmid stability 34,35,55,91 . However, the location of the Thr69Ile missense change at the binding interface between Mms21 and Smc5 (Supplementary Figure S9) suggested an alternate mechanism that relies on the Smc5/6 complex.…”