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

Deena Jalal,Jisha Chalissery,Ahmed H Hassan

doi:10.3389/fmolb.2020.00221

Deena Jalal, Jisha Chalissery + Show 1 more

Open Access

https://doi.org/10.3389/fmolb.2020.00221

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Abstract

The endogenous yeast 2-μm plasmid while innocuous to the host, needs to be properly regulated to avoid a toxic increase in copy number. The plasmid copy number control system is under the control of the plasmid encoded recombinase, Flp1. In case of a drop in 2-μm plasmid levels due to rare plasmid mis-segregation events, the Flp1 recombinase together with the cell’s homologous recombination machinery, produce multiple copies of the 2-μm plasmid that are spooled during DNA replication. The 2-μm plasmid copy number is tightly regulated by controlled expression of Flp1 as well as its ubiquitin and SUMO modification. Here, we identify a novel regulator of the 2-μm plasmid, the ATPase, ubiquitin ligase, Irc20. Irc20 was initially identified as a homologous recombination regulator, and here we uncover a new role for Irc20 in maintaining the 2-μm plasmid copy number and segregation through regulating Flp1 protein levels in the cell.

Highlights

The Saccharomyces cerevisiae 2-μm plasmid is an endogenous selfish circular plasmid that utilizes the cell’s own machinery to maintain copy number at 40–60 copies per haploid cell and segregation efficiency close to that of chromosomes
We show that mutations in irc20 frequently caused the loss of the otherwise highly stable 2-μm plasmid, in a manner dependent on Flp1 expression, and not through the control of the Rep1-Rep2-STB pathway
In addition to a role in plasmid segregation, we show that despite the lack of cell sensitivities associated with increased 2-μm plasmid levels, irc20 mutant increased 2-μm plasmid levels 4-folds compared to the WT

Summary

Introduction

The Saccharomyces cerevisiae 2-μm plasmid is an endogenous selfish circular plasmid that utilizes the cell’s own machinery to maintain copy number at 40–60 copies per haploid cell and segregation efficiency close to that of chromosomes (reviewed in Chan et al, 2013; Liu et al, 2014). The faithful segregation of the 2-μm plasmid to the daughter cells is coordinated by the plasmid partitioning system which comprises two plasmid encoded proteins, Rep and Rep and a cis-acting DNA element STB (Jayaram et al, 2004). Rep and Rep proteins form a complex at the STB locus, which acts in a manner similar to yeast centromeres where it recruits other host-encoded factors, such as Rsc and the cohesin complex (Wong et al, 2002; Yang et al, 2004), and helps in segregating the replicated plasmid into the mother and daughter cells. In rare mis-segregation events of plasmid molecules, the plasmid encoded recombinase Flp corrects the copy number by inducing a cut at the Flp recognition target (FRTs) initiating a double rolling circle replication mode which amplifies copy number with the help of the cellular homologous recombination (HR) machinery (Futcher and Cox, 1983; Futcher, 1986). The activity of Flp is tightly regulated at the level of gene expression through Rep1-Rep mediated inhibition (Murray et al, 1987), and by SUMO modification to regulate its

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