Yeast Stn1 promotes MCM to circumvent Rad53 control of the S phase checkpoint

Hovik Gasparayan,Jeff Bachant,Chris Caridi,Wenyi Feng,Jeff Julius,Constance I Nugent

doi:10.1007/s00294-022-01228-0

Hovik Gasparayan, Jeff Bachant + Show 4 more

Open Access

https://doi.org/10.1007/s00294-022-01228-0

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Abstract

Treating yeast cells with the replication inhibitor hydroxyurea activates the S phase checkpoint kinase Rad53, eliciting responses that block DNA replication origin firing, stabilize replication forks, and prevent premature extension of the mitotic spindle. We previously found overproduction of Stn1, a subunit of the telomere-binding Cdc13–Stn1–Ten1 complex, circumvents Rad53 checkpoint functions in hydroxyurea, inducing late origin firing and premature spindle extension even though Rad53 is activated normally. Here, we show Stn1 overproduction acts through remarkably similar pathways compared to loss of RAD53, converging on the MCM complex that initiates origin firing and forms the catalytic core of the replicative DNA helicase. First, mutations affecting Mcm2 and Mcm5 block the ability of Stn1 overproduction to disrupt the S phase checkpoint. Second, loss of function stn1 mutations compensate rad53 S phase checkpoint defects. Third Stn1 overproduction suppresses a mutation in Mcm7. Fourth, stn1 mutants accumulate single-stranded DNA at non-telomeric genome locations, imposing a requirement for post-replication DNA repair. We discuss these interactions in terms of a model in which Stn1 acts as an accessory replication factor that facilitates MCM activation at ORIs and potentially also maintains MCM activity at replication forks advancing through challenging templates.

Highlights

Cells must tolerate various forms of DNA replication stress, ranging from extrinsic mutagens to endogenous physiological perturbations
To identify genetic pathways affected by STN1 overproduction (STN1 OP), we chose an approach based on our previous finding that STN1 OP checkpoint defects could be suppressed in a pol12–40 mutant (Gasparyan et al 2009)
The Pol12–40 mutant protein is partially defective for binding Stn1 (Petreaca et al 2006; see schematic of Stn1 domains and interactions, Fig. 1A), and retention of excess Stn1 on chromatin spreads is greatly reduced in pol12–40 mutants (Gasparyan et al 2009), implying STN1 OP acts through POL12

Summary

Introduction

Cells must tolerate various forms of DNA replication stress, ranging from extrinsic mutagens to endogenous physiological perturbations. Current Genetics mitotic spindle that assembles in HU-arrested yeast cells (Krishnan et al 2004; Bachant et al 2005; Julius et al 2019) These responses synergize to allow DNA synthesis to proceed at a slow but steady rate in HU (Alvino et al 2007; Poli et al 2012; Zhong et al 2013), to circumvent dNTP depletion (Morafraile et al 2015), and to retain the capacity for accurate chromosome segregation once the extended S phase has been completed (Feng et al 2009). Unregulated Exo activity in rad mutants generated CEN ssDNA and perturbed kinetochore assembly Based on these findings, we proposed that the critical role for Rad in restraining spindle extension in HU is to stabilize replication forks in proximity to CENs. In the absence of this protective function, exonucleolytic degradation of CEN DNA disrupts kinetochore integrity and S phase spindle force balancing mechanisms. Our results indicate Stn is likely to act in concert with both the DDK and the MCM complex to efficiently activate ORI firing-a function revealed most prominently when ORIs fire in an unscheduled manner in the absence of the S phase checkpoint

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