Budding Yeast Rif1 Controls Genome Integrity by Inhibiting rDNA Replication.

Maksym Shyian,Michael Costanzo,Aleksandra Lezaja,Stefano Mattarocci,Charlie Boone,David Shore,Benjamin Albert,Lukas Hafner,Dana Branzei

doi:10.1371/journal.pgen.1006414

Maksym Shyian, Michael Costanzo + Show 7 more

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https://doi.org/10.1371/journal.pgen.1006414

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Journal: PLoS Genetics	Publication Date: Nov 7, 2016
Citations: 31	License type: CC BY 4.0

Affiliation: University of Geneva, University of Toronto

Abstract

The Rif1 protein is a negative regulator of DNA replication initiation in eukaryotes. Here we show that budding yeast Rif1 inhibits DNA replication initiation at the rDNA locus. Absence of Rif1, or disruption of its interaction with PP1/Glc7 phosphatase, leads to more intensive rDNA replication. The effect of Rif1-Glc7 on rDNA replication is similar to that of the Sir2 deacetylase, and the two would appear to act in the same pathway, since the rif1Δ sir2Δ double mutant shows no further increase in rDNA replication. Loss of Rif1-Glc7 activity is also accompanied by an increase in rDNA repeat instability that again is not additive with the effect of sir2Δ. We find, in addition, that the viability of rif1Δ cells is severely compromised in combination with disruption of the MRX or Ctf4-Mms22 complexes, both of which are implicated in stabilization of stalled replication forks. Significantly, we show that removal of the rDNA replication fork barrier (RFB) protein Fob1, alleviation of replisome pausing by deletion of the Tof1/Csm3 complex, or a large deletion of the rDNA repeat array all rescue this synthetic growth defect of rif1Δ cells lacking in addition either MRX or Ctf4-Mms22 activity. These data suggest that the repression of origin activation by Rif1-Glc7 is important to avoid the deleterious accumulation of stalled replication forks at the rDNA RFB, which become lethal when fork stability is compromised. Finally, we show that Rif1-Glc7, unlike Sir2, has an important effect on origin firing outside of the rDNA locus that serves to prevent activation of the DNA replication checkpoint. Our results thus provide insights into a mechanism of replication control within a large repetitive chromosomal domain and its importance for the maintenance of genome stability. These findings may have important implications for metazoans, where large blocks of repetitive sequences are much more common.

Highlights

In eukaryotes DNA replication initiates from multiple sites in a characteristic sequential pattern referred to as ‘replication timing’ [1]
Rif1 is a conserved eukaryotic protein implicated in regulation of both the temporal pattern of DNA replication initiation and the DNA damage response (DDR)
Our results show that inhibition of rDNA origin firing by Rif1-Glc7/phosphatase 1 (PP1) prevents the buildup of an excess of stalled forks within the rDNA locus, which can lead to genome instability and cell death

Summary

Introduction

In eukaryotes DNA replication initiates from multiple sites (origins) in a characteristic sequential pattern referred to as ‘replication timing’ [1]. Replication timing is tightly regulated, with some origins being replicated early during S phase and others later [2, 3]. In the budding yeast Saccharomyces cerevisiae about one-third of all potential replication origins (autonomously replicating sequences, or ARSs) are located within the rDNA repeat array on chromosome XII [6]. The rDNA array comprises ~150–200 copies of a module containing 35S and 5S rRNA genes separated by two intergenic regions harboring an origin of replication (rARS) and a polar replication fork barrier (RFB) (Fig 1A). Only ~20% of the rDNA origins fire during a given S phase in wild type cells [6, 7], and it has been shown that deregulation of rDNA replication leads to genomic instability [8, 9]

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