S phase

Abstract: The cyclin-dependent kinase (Cdk) enzymes, when associated with the G1 cyclins D and E, are rate-limiting for entry into the S phase of the cell cycle. During T-cell mitogenesis, antigen-receptor signalling promotes synthesis of cyclin E and its catalytic partner, Cdk2, and interleukin-2 (IL-2) signalling activates cyclin E/Cdk2 complexes. Rapamycin is a potent immunosuppressant which specifically inhibits G1-to-S-phase progression, leading to cell-cycle arrest in yeast and mammals. Here we report that IL-2 allows Cdk activation by causing the elimination of the Cdk inhibitor protein p27Kip1, and that this is prevented by rapamycin. By contrast, the Cdk inhibitor p21 is induced by IL-2 and this induction is blocked by rapamycin. Our results show that p27Kip1 governs Cdk activity during the transition from quiescence to S phase in T lymphocytes and that p21 function may be restricted to cycling cells.

Abstract: The DNA damage response pathway is a cellular surveillance system that senses the presence of damaged DNA and elicits an appropriate and effective response to that damage. First identified as a regulator of cell cycle transitions, the DNA damage response pathway has since been shown to regulate a number of other cellular processes, which include DNA repair, apoptosis, and replication fork stabilization (Zhou and Elledge 2000; Cimprich 2003). The importance of this pathway is demonstrated by the conservation of this response from yeast to humans (Zhou and Elledge 2000; Melo and Toczyski 2002) and by several studies that have shown that loss of checkpoint proteins predisposes affected individuals to cancer (Sherr 2004). One critical component of the DNA damage response pathway is the ATR–ATRIP complex. ATR is a phosphatidylinositol kinase-related protein kinase that is thought to function as both a sensor and transducer in the DNA damage response. ATR, and its associated protein ATRIP, respond to a broad spectrum of genotoxic agents that includes ultraviolet light (UV), topoisomerase inhibitors, alkylating agents, and cis-platinum, as well as chemicals that disrupt replication, such as aphidicolin and hydroxyurea (HU) (Zhou and Elledge 2000; Cortez et al. 2001; Melo and Toczyski 2002). Following DNA damage, ATR phosphorylates and activates the checkpoint kinase Chk1 (Melo and Toczyski 2002). In higher eukaryotes, the phosphorylation of Chk1 also requires the activities of the Rad9–Rad1–Hus1 (RHR, aka 9–1–1) complex and Claspin (Melo and Toczyski 2002). The RHR complex is a PCNA-related complex that is loaded on to primed DNA in vitro (Ellison and Stillman 2003; Zou et al. 2003) and is recruited to sites of DNA damage in vivo (Kondo et al. 2001; Melo et al. 2001). Claspin was initially identified as a protein that bound the activated form of Chk1, and it has been shown to bind chromatin throughout S phase (Kumagai and Dunphy 2000; Lee et al. 2003). We and others previously showed that recruitment of ATR and Rad1 to chromatin (Lupardus et al. 2002) and activation of Chk1 (Lupardus et al. 2002; Stokes et al. 2002) requires DNA replication in Xenopus egg extracts following several types of DNA damage. Studies in mammalian cells also indicate that ATR binds UV-damaged chromatin in S phase but not G1 phase (Ward et al. 2004). In addition, other studies show that a replication fork must be established in Saccharomyces cerevisiae for checkpoint activation induced by methylmethane sulfonate (MMS) (Tercero et al. 2003). Taken together, these observations suggest that one or more replication-dependent events are needed to generate the signal that ATR recognizes for many types of DNA damage. Although the exact nature of the biochemical signal(s) responsible for activating the ATR pathway and the replication-dependent steps necessary for its formation are still unclear, evidence from a number of different systems supports a central role for replication protein A (RPA)-coated single-stranded DNA (ssDNA) in the response. In yeast, certain RPA mutants exhibit a checkpoint defect and also adapt more rapidly to DNA damage (Longhese et al. 1996; Pellicioli et al. 1999). In addition, knock-down of the ssDNA-binding protein RPA results in a significant loss of both Chk1 phosphorylation and ATR foci formation following DNA damage in mammalian cells (Zou and Elledge 2003). In Xenopus egg extracts, RPA is also required for the recruitment of ATR to chromatin following treatment with aphidicolin (You et al. 2002) or etoposide (Costanzo et al. 2003) and for the recruitment of ATR to poly(dA)70 ssDNA (Lee et al. 2003). Importantly, in vitro experiments have shown that RPA is sufficient for the binding of ATRIP to ssDNA (Zou and Elledge 2003) and that RPA also facilitates the association of the RHR complex with DNA (Ellison and Stillman 2003; Zou et al. 2003). Interestingly, the amount of ssDNA appears to increase following genotoxic stress, as RPA accumulates on chromatin in Xenopus extracts and mammalian cells treated with UV, MMS, HU, or aphidicolin (Michael et al. 2000; Mimura et al. 2000; Walter 2000; Lupardus et al. 2002; Zou and Elledge 2003). Moreover, in budding yeast, increased amounts of ssDNA have been observed by electron microscopy following HU treatment (Sogo et al. 2002). In the case of DNA damage, the mechanism by which this ssDNA accumulates is not known, nor is it clear if ssDNA accumulation is required for checkpoint activation. In principle, a number of DNA repair (e.g., nucleotide excision repair, base excision repair) and recombination processes could lead to the generation of ssDNA following DNA damage at several points in the cell cycle. Alternatively, during DNA replication, ssDNA could be formed if DNA polymerases are slowed by lesions and the replicative helicase continues to unwind DNA. Indeed, uncoupling of helicase and polymerase activities has been previously observed in the presence of aphidicolin (Walter and Newport 2000), and recent studies have shown that this aphidicolin-induced uncoupling is dependent on the MCM helicase (Pacek and Walter 2004). In this study, we used a cell-free extract system derived from Xenopus eggs (Walter et al. 1998) to examine the mechanism by which ssDNA accumulates following DNA damage. We demonstrate that the appearance and disappearance of a highly unwound form of plasmid DNA that accumulates following aphidicolin treatment (Walter and Newport 2000) correlates with the phosphorylation of Chk1 on Ser 344 (S344). Importantly, this hyperunwound form of DNA was also observed upon replication of plasmid DNA damaged with either UV or cis-platinum. This suggests that DNA damage induces uncoupling of helicase and polymerase activities and that these lesions, as well as aphidicolin, may generate a common checkpoint-activating DNA structure. Moreover, while stalling the replication fork with aphidicolin results in a robust checkpoint response, we find that aphidicolin elicits no checkpoint when the MCM DNA helicase is inactivated. Using plasmids of varying sizes, we also show that functional uncoupling of DNA unwinding and DNA synthesis during S phase may serve to amplify the level of Chk1 phosphorylation that can be achieved at each individual replication fork. Finally, we demonstrate that although DNA unwinding is necessary for checkpoint activation, it is not sufficient and that additional DNA synthesis by Polα is needed. Taken together, these results suggest that functional uncoupling of helicase and polymerase activities is necessary to convert DNA lesions and chemical inhibitors of DNA replication into the signal(s) that activate the ATR-dependent checkpoint.

Abstract: In eukaryotes a cell-cycle control termed a checkpoint causes arrest in the S or G2 phases when chromosomes are incompletely replicated or damaged. Previously, we showed in budding yeast that RAD9 and RAD17 are checkpoint genes required for arrest in the G2 phase after DNA damage. Here, we describe a genetic strategy that identified four additional checkpoint genes that act in two pathways. Both classes of genes are required for arrest in the G2 phase after DNA damage, and one class of genes is also required for arrest in S phase when DNA replication is incomplete. The Gz-specific genes include MEC3 (for mitosis entry checkpoint), RAD9, RAD17, and RAD24. The genes common to both S phase and G2 phase pathways are MECl and MEC2. The MEC2 gene proves to be identical to the RAD53 gene. Checkpoint mutants were identified by their interactions with a temperature-sensitive allele of the cell division cycle gene CDC13-, cdcl3 mutants arrested in G2 and survived at the restrictive temperature, whereas all cdcl3 checkpoint double mutants failed to arrest in G2 and died rapidly at the restrictive temperature. The cell-cycle roles of the RAD and MEC genes were examined by combination of rad and mec mutant alleles with 10 cdc mutant alleles that arrest in different stages of the cell cycle at the restrictive temperature and by the response of rad and mec mutant alleles to DNA damaging agents and to hydroxyurea, a drug that inhibits DNA replication. We conclude that the checkpoint in budding yeast consists of overlapping S-phase and G2-phase pathways that respond to incomplete DNA replication and/or DNA damage and cause arrest of cells before mitosis.