Checkpoint-independent scaling of the Saccharomyces cerevisiae DNA replication program

Abstract

Background: In budding yeast, perturbations that prolong S phase lead to a proportionate delay in the activation times of most origins. The DNA replication checkpoint was implicated in this scaling phenotype, as an intact checkpoint was shown to be required for the delayed activation of late origins in response to hydroxyurea treatment. In support of that, scaling is lost in cells deleted of mrc1, a mediator of the replication checkpoint signal. Mrc1p, however, also plays a role in normal replication. Results: To examine whether the replication checkpoint is required for scaling the replication profile with S phase duration we measured the genome-wide replication profile of different MRC1 alleles that separate its checkpoint function from its role in normal replication, and further analyzed the replication profiles of S phase mutants that are checkpoint deficient. We found that the checkpoint is not required for scaling; rather the unique replication phenotype of mrc1 deleted cells is attributed to the role of Mrc1 in normal replication. This is further supported by the replication profiles of tof1Δ which functions together with Mrc1p in normal replication, and by the distinct replication profiles of specific POL2 alleles which differ in their interaction with Mrc1p.

Cell-to-cell variability and robustness in S-phase duration from genome replication kinetics

“…Interestingly, our predictions of S-phase duration and variability as a function of chromosome copy numbers ( Supplementary Figure S12 ) might apply to cancer cell lines with different levels of aneuploidy ( 37 ). Finally, there is the possibility of applying this framework to describe relevant perturbations ( 40 , 41 ). This could also help elucidate how response to DNA damage affects the replication timing and its variability across cells.…”

Model-based analysis of DNA replication profiles: predicting replication fork velocity and initiation rate by profiling free-cycling cells

“…It is less well suited for characterizing wild-type cells, primarily because the SVD projections are not intuitive and do not provide explicit information about the parameters controlling replication. Curve-fitting methods that attempt to more directly model the full replication profile are better suited for characterizing the wild-type profiles and were indeed applied to multiple systems ranging from a Xenopus embryo (Goldar et al 2008;Yang and Bechhoefer 2008;Gauthier and Bechhoefer 2009), budding yeast (Brümmer et al 2010;de Moura et al 2010;Yang et al 2010;Retkute et al 2011), regions of human cells (Gindin et al 2014), and others (Hyrien and Goldar 2010;Koutroumpas and Lygeros 2011;Retkute et al 2012;Baker and Bechhoefer 2014). These methods are labor-intensive and require an expert user to set up.…”

“…First, we considered cells deleted of MRC1 or RIF1, which were predicted to reduce fork velocity based on their shorter replicon length and longer S phase relative to wild type. Mrc1 was previously shown to promote fork velocity (Szyjka et al 2005;Tourriere et al 2005;Hodgson et al 2007;Gispan et al 2014), while Rif1 was implicated in regulating telomeric origins (Hayano et al 2012;Yamazaki et al 2013;Peace et al 2014). Second, we considered the clb5Δ and fkh 1,2 ΔΔ mutants, predicted to respectively increase or decrease initiation rate (Donaldson et al 1998;Knott et al 2012): clb5Δ, a B-type cyclin, increased replicon length and S phase duration, while fkh 1,2 ΔΔ decreased replicon length but did not increase S phase.…”

Cdc48 targets INQ-localized Mrc1 to facilitate recovery from replication stress

“…Finally, recognition by Cdc48-associated Otu1 may result in the removal of ubiquitin modifications from Mrc1 (Rumpf & Jentsch, 2006;Stein et al, 2014). Since Mrc1 is required for efficient replication in unperturbed cells (Gispan et al, 2014;Hodgson et al, 2007;Szyjka et al, 2005), de-modification of Mrc1 may rescue the protein from proteasomal degradation and facilitate Cdc48-mediated extraction of Mrc1 from INQ. De-modified Mrc1 could thus return to the replication fork during recovery from MMS-induced replication stress in order to efficiently restart and complete replication.…”

“…Apart from Mrc1, the fork protection complex consists of the Csm3 and Tof1 proteins, both of which are required for Mrc1 presence at the replication fork (Bando et al, 2009;Uzunova et al, 2014). Finally, Mrc1 is required at the replication fork to promote efficient DNA synthesis during unperturbed replication (Gispan et al, 2014;Hodgson et al, 2007;Szyjka et al, 2005;Yeeles et al, 2017).…”