Mrc1 and Tof1 Promote Replication Fork Progression and Recovery Independently of Rad53

Abstract

The yeast checkpoint factors Mrc1p and Tof1p travel with the replication fork and mediate the activation of the Rad53p kinase in response to a replication stress. We show here that both proteins are required for normal fork progression but play different roles at stalled forks. Tof1p is critical for the activity of the rDNA replication fork barrier (RFB) but plays a minor role in the replication checkpoint. In contrast, Mrc1p is not necessary for RFB activity but is essential to mediate the replication stress response. Interestingly, stalled forks did not collapse in mrc1Delta cells exposed to hydroxyurea (HU) as they do in rad53 mutants. However, forks failed to restart when mrc1Delta cells were released from the block. The critical role of Mrc1p in HU is therefore to promote fork recovery in a Rad53p-independent manner, presumably through the formation of a stable fork-pausing complex.

Defining replication origin efficiency using DNA fiber assays

“…Importantly, DNA combing studies also revealed the presence of large inactive regions in HU-treated cells (Tourriere et al 2005;Versini et al 2003). These domains are absent in checkpoint mutants, indicating that they correspond to latereplicating regions repressed by checkpoint kinases (Tourriere et al 2005). Although origin usage is extremely flexible within early replication domains (Fig.…”

“…In initial DNA combing experiments, fiber lengths could not be determined for technical reasons, and IODs were essentially measured within clusters of early origins (Lengronne et al 2001;Lengronne and Schwob 2002;Shimada et al 2002). With the development of single-stranded DNA (ssDNA) staining procedures, BrdU tracks could be assigned to the same fiber over much longer distances, revealing the presence of large repressed domains in between clusters of active origins (Tourriere et al 2005). IODs measured after counterstaining of DNA fibers are 25% larger for the same cells (Fig.…”

“…Cells were then arrested in G 1 of the next cell cycle and were further released into S phase for another 90 min in the presence of CldU (green) and HU. DNA fibers were stretched by DNA combing and halogenated nucleotides were detected with specific antibodies as described (Tourriere et al 2005). As shown in Fig.…”

“…Mrc1 and Tof1 form a complex with Csm3 at the replication fork and have been implicated in the coordination between replicative helicases and polymerases (Katou et al 2003). When mrc1Δ and tof1Δ mutants are exposed to HU, replisome components uncouple from sites of DNA synthesis (Katou et al 2003) and forks are unable to restart after release from HU (Tourriere et al 2005). Yet, tof1Δ mutants are much less sensitive to HU than mrc1Δ cells.…”

Ccr4 contributes to tolerance of replication stress through control ofCRT1mRNA poly(A) tail length

“…The acute sensitivity of the ccr4⌬ dun1⌬ strain suggested that the mutant may be defective in stabilization of DNA replication forks, a phenotype observed in mec1 or rad53 checkpoint mutants (Lopes et al, 2001;Tercero and Diffley, 2001). We therefore examined whether ccr4⌬ dun1⌬ strains were irreversibly sensitive to HU (Allen et al, 1994), a phenotype associated with replication fork collapse (Branzei and Foiani, 2005;Tourriere et al, 2005). Each of the wild-type and single mutant strains tolerated an acute exposure to HU and formed colonies on medium lacking drug, whereas less than 1% of the ccr4⌬ dun1⌬ starting cell population was viable after exposure to HU for a 24-hour period, indicating irreversible HU sensitivity (Fig.…”