Checkpoint Signaling in Yeast
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Checkpoint Signaling in Yeast
The maintenance of genomic integrity is important for the survival of eukaryotic cells. Checkpoint mechanisms prevent cell cycle transitions until previous events have been completed or damaged DNA has been repaired. Various forms of DNA damage and various treatments that block replication trigger these checkpoints. Checkpoint pathways and proteins are evolutionarily conserved from yeast to man, underlining their importance in maintaining genomic integrity. In the fission yeast, Schizosaccharomyces pombe, several checkpoint pathways monitor the status of the DNA and arrest the cell cycle in response to DNA damage or inhibition of DNA replication induced by Ionizing Radiation and UV light (Ref.1). The checkpoint Rad proteins, Rad1, Rad3, Rad4, Rad9, Rad17, Rad26 and Hus1, are required for damage sensing and processing and are involved both in the DNA damage checkpoint pathway and in responding to replication arrest.

In S. pombe checkpoint proteins interact with the RC (Replication Complex) during S-phase or with DNA strand breaks if they are induced during G2-phase. These checkpoint proteins ultimately inactivate the CDC2-Cyclin-B kinase complex by inhibitory Tyr15 phosphorylation (Ref.2). High CDC2-Cyclin-B kinase activity allows the cell to enter mitosis, where chromosome separation and segregation occur. Checkpoint activation thus causes cell cycle arrest in G2-phase. There are two known effector kinases downstream of the checkpoint Rad proteins, Chk1 and Cds1. Chk1 is phosphorylated in response to DNA damage induced in late S or G2 in a Rad3-dependent manner, whereas Cds1 is activated as a part of the intra-S checkpoint when the DNA is damaged during S-phase or when DNA replication is inhibited by hydroxyurea or by a DNA Polymerase defect (Ref.3). Activation of either kinase leads to inhibition of CDC2 activity by maintaining the inhibitory phosphorylation of Tyr15. Tyr15 of CDC2 is phosphorylated by Wee1 and Mik1, and dephosphorylated in preparation for mitosis by CDC25. The CDC2p has a major role in controlling cell cycle progression through both the G1/S and G2/M transitions. At the onset of M-phase, dephosphorylation of the Tyr15 residue of CDC2p leads to kinase activation and is carried out by the CDC25p phosphatase. At entry into S-phase, CDC2p forms a complex with another B-type Cyclin encoded by the Cig2 gene and promotes passage through the start control point.

The Rad3-Rad26 (ATR-ATRIP complex) phosphorylates many of the other checkpoint proteins, and these events are central to the checkpoint response. Rad3-dependent Chk1 and Cds1 phosphorylation is essential for checkpoint activation (Ref.4). Rad17, together with RFC2-RFC5, forms an RFC-like complex and loads a PCNA (Proliferating-Cell Nuclear Antigen)-like Rad1-Hus1-Rad9 complex onto damaged DNA. This complex links Rad3-Rad26 to the mediator complexes, which in turn bring Chk1 and Cds1 into proximity. Different mediator complexes function in S-phase and G2-phase. In S-phase, Mrc1 mediates Cds1-dependent checkpoint signaling. Activation of Cds1 kinase results in Mik1 accumulation. Mik1 phosphorylates Tyr15 of CDC2, thus preventing mitosis. Importantly, the Mrc1/Cds1 module has additional roles in the S-phase response, such as stabilizing stalled replication forks and inhibiting late-firing replication origins. It also regulates the Mus81-Eme1 Endonuclease complex (Ref.5). In G2-phase, mediator protein Crb2 mediates Chk1-dependent checkpoint signaling through 14-3-3. Activated Chk1 results in activation of Mik1 and Wee1 and may inactivate CDC25 tyrosine phosphatase. Cds1 also phosphorylates for the 14-3-3 families of proteins on Ser216, the consensus-binding site for 14-3-3 that regulate biochemical activities by binding to and sequestering phosphorylated proteins. The CDC25C-14-3-3 interaction may prevent mitosis by sequestering CDC25C in the cytoplasm, where it cannot dephosphorylate and activate CDC2-Cyclin-B complexes.

Chk1 is also involved in regulating dNTP synthesis. Chk1 activation in G2 induces degradation of the Spd1 (for S-phase delayed) protein. Spd1 behaves as an inhibitor of RNR (Ribonucleotide Reductase). Its Ubiquitin-dependent degradation is necessary for nuclear export of Suc22 (the small subunit of RNR) during DNA replication in S-phase and DNA repair in G2-phase (Ref.6). Suc22 , which normally localizes to the nucleus in G2-phase cells and the whole cell during normal S-phase, presumably forms an active complex with cytoplasmic CDC22 during DNA replication and in response to checkpoint activation to provide nucleotides for DNA repair. The signalosome complex promotes Ubiquitin-dependent degradation of Spd1. The signalosome forms a complex together with the Pcu4 (Cullin-4) E3 Ubiquitin Ligase (Ref.6).

In addition to the targets discussed above, there are other targets of Chk1 and Cds1. Cds1 is required to maintain cell viability during a replication arrest, which is independent of its role in preventing mitosis (Ref.7). Fission yeast cells spend most of their time in G2, and the DNA damage and replication checkpoints prevent cell cycle progression by blocking the G2/M transition by inhibiting the CDC2 kinase (Ref.4).