ORC and the intra-S-phase checkpoint: a threshold regulates Rad53p activation in S phase

Abstract

The intra-S-phase checkpoint in yeast responds to stalled replication forks by activating the ATM-like kinase Mec1 and the CHK2-related kinase Rad53, which in turn inhibit spindle elongation and late origin firing and lead to a stabilization of DNA polymerases at arrested forks. A mutation that destabilizes the second subunit of the Origin Recognition Complex, orc2-1, reduces the number of functional replication forks by 30% and severely compromises the activation of Rad53 by replication stress or DNA damage in S phase. We show that the restoration of the checkpoint response correlates in a dose-dependent manner with the restoration of pre-replication complex formation in G1. Other forms of DNA damage can compensate for the reduced level of fork-dependent signal in the orc2-1 mutant, yet even in wild-type cells, the amount of damage required for Rad53 activation is higher in S phase than in G2. Our data suggest the existence of an S-phase-specific threshold that may be necessary to allow cells to tolerate damage-like DNA structures present at normal replication forks. As cells proliferate, the genome must be replicated and segregated with high fidelity. Surveillance mechanisms, or checkpoints, detect damaged DNA or on-going replication and block cell cycle progression in order to allow adequate time to repair the damaged DNA or to complete replication (Weinert 1998;Zhou and Elledge 2000). Failure to delay the cell cycle can convert an easily reparable DNA lesion into one more deleterious, resulting in chromosomal rearrangements or loss. The importance of this mechanism is underscored by a large number of heritable human diseases that arise from defects in checkpoint or DNA-damage repair functions (for review, see Kastan 1997).Genetic analysis of the DNA damage checkpoint pathway in yeast has allowed classification of its components into sensors, which detect DNA damage, adaptors, which integrate and transmit the signal, and effector kinases, which promote downstream functions including the induction of repair genes, suppression of cell cycle progression, the arrest of replication polymerases, downregulation of late origins and of sister chromatid segregation (for review, see Zhou and Elledge 2000; Melo and Toczyski 2002). In G1 and G2 phases of the budding yeast cell cycle, a DNA-bound complex of Rad17/Mec3/ Ddc1 and Rad24 proteins, as well as a complex containing the ATM homolog Mec1p and its cofactor Ddc2p, play crucial roles as sensors. These communicate through Rad9p to activate the major effector kinases, Rad53p (CHK2 in other species) and Chk1p. These components and their roles are conserved from yeast to man (Melo and Toczyski 2002).The response of the cell to DNA damage or fork arrest during S phase is distinct from the G1/S or G2/M checkpoints, both at the level of sensors and with respect to the cellular response to Rad53p activation. It is useful to consider this intra-S-phase response with respect to two operationally distinct lesions. The first arises from replication forks stalled by high concentra...

Defining replication origin efficiency using DNA fiber assays

“…These authors were also able to reveal subtle initiation defects in cells defective for the CDK inhibitor Sic1, which were hardly detectable with classical biochemical assays (Lengronne and Schwob 2002). Stronger initiation defects were also measured in various initiation mutants using this approach (Devault et al 2002;Semple et al 2006;Shimada et al 2002). .…”

“…2D gel analyses and microarray data show that only a subset of these ARS elements is active on the chromosome (1/35 kb) (Newlon and Theis 1993;Raghuraman et al 2001). DNA combing analyses indicate that only a fraction of chromosomal origins are active in a given cell (1/46 kb) (Lengronne et al 2001;Lengronne and Schwob 2002;Shimada et al 2002). Analysis of larger DNA fibers by DNA combing reveals the presence of large unreplicated domains containing late origins, increasing again the average IOD (1/58 kb).…”

“…3a, interorigin distances (IODs) are influenced by the overall length of analyzed DNA fibers. 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).…”

Suppression of replication fork progression in low-dose-specific p53-dependent S-phase DNA damage checkpoint

“…The phosphorylation and focus formation of PCNA, and the suppression of DNA synthesis were p53-dependent at doses below 2.5 Gy, but not at higher doses, suggesting the damage level dependence of the S-phase DNA damage checkpoint. In fact, the S-phase DNA damage checkpoint was reported to require a threshold level of damage to activate the checkpoint response (Shimada et al, 2002). Full activation of Chk2, an effector of the S-phase DNA damage checkpoint, was reported to require radiation doses above 2 Gy (Buscemi et al, 2004), indicating the presence of a threshold for the induction of the whole battery of S-phase DNA damage responses.…”

A Conditional Yeast E1 Mutant Blocks the Ubiquitin–Proteasome Pathway and Reveals a Role for Ubiquitin Conjugates in Targeting Rad23 to the Proteasome

“…A second allele resulting from a transposon insertion upstream of the UBA1 coding sequence reduced wild-type Uba1 protein function, causing inefficient degradation of some proteins (Swanson and Hochstrasser, 2000). Additional alleles were later encountered as suppressor mutations in various indirect genetic screens, and they also achieved only partial inactivation of the pathway (Cheng et al, 2002;Shimada et al, 2002). Whereas these hypomorphic alleles have proven useful in confirming the ubiquitin dependence of the turnover of specific proteins, they highlight the potential value of a stronger conditional allele with broad utility in exploring the entire ubiquitin conjugation pathway.…”