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

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. Author summary Plasmids of budding yeasts, exemplified by the 2-micron plasmid of Saccharomyces cerevisiae, and mammalian papilloma and gammaherpes viruses typify eukaryotic extra-chromosomal selfish DNA elements. The plasmid and the viral episomes, despite the long evolutionary divergence of their hosts, share striking similarities in lifestyles. These include the ability to segregate to daughter cells by hitchhiking on chromosomes and to switch from cell cycle regulated replication to iterative replication for copy number maintenance. While selfish elements, including those integrated into chromosomes, rely on their hosts' genetic potential for long-term survival, their genetic load is carefully regulated to minimize fitness conflicts with the hosts. Our study focuses on the Flp site-specific recombinase, which is central to the copy number control of the 2-micron plasmid and whose steady state levels are regulated through transcriptional control by plasmid coded proteins and through post-translational modification by the host's sumoylation machinery. We demonstrate that sumoylation, in addition, attenuates the catalytic activity of Flp by diminishing its DNA binding affinity and inter-monomer cooperativity, providing another layer of protection against runaway increase in plasmid copy number. Population control by self-imposed and host-mediated mechanisms is likely a general strategy among selfish elements to ensure nearly conflict-free coexistence with host genomes.