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

Chien-Hui Ma,Piotr Guga,Hsiu-Fang Fan,Anna Maciaszek,Bo-Yu Su,Makkuni Jayaram,Harmit S Malik

doi:10.1371/journal.pgen.1008193

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.

Highlights

The yeast 2-micron plasmid, nearly ubiquitous among Saccharomyces yeast strains, is a highly optimized extrachromosomal selfish DNA element [1,2,3,4]
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
The partitioning system [5,6,7], comprised of the plasmid-coded Rep1 and Rep2 proteins together with a cis-acting locus STB, promotes nearly equal segregation of plasmid molecules duplicated by the host replication machinery into mother and daughter cells

Summary

Introduction

The yeast 2-micron plasmid, nearly ubiquitous among Saccharomyces yeast strains, is a highly optimized extrachromosomal selfish DNA element [1,2,3,4]. Current evidence is consistent with a ‘hitchhiking model’ in which the plasmid utilizes chromosomes as a vehicle for segregation by physically associating with them [8,9,10,11]. In this respect, the plasmid resembles the episomes of mammalian papilloma and gammaherpes viruses that resort to chromosome-tethering for stable maintenance during prolonged periods of latent infection [12,13,14,15,16,17,18,19,20]. It is possible that selfish genomes inhabiting evolutionarily distant hosts have independently converged on the common strategy of chromosome-coupled segregation as a means for self-preservation

Methods

Results

Discussion

Conclusion