Checkpoint Comparative Pathways
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Checkpoint Comparative Pathways

Checkpoints monitor critical cell cycle events such as chromosome duplication and segregation. They are highly conserved mechanisms that prevent progression into the next phase of the cell cycle when cells are unable to accomplish the previous event properly. The DNA replication checkpoint is crucial for maintaining genome stability, because replication forks become vulnerable to collapse when they encounter obstacles such as nucleotide adducts nicks, RNA-DNA hybrids, or stable protein-DNA complexes. The DNA damage checkpoint arrests cell cycle progression to allow time for repair. Once DNA repair is completed, checkpoint signaling is terminated (Ref.1 and 2)

The first step in the initiation of activity of DNA damage checkpoints is recognition of the DNA damage. Studies in yeasts and mammals have demonstrated that Rad9, Rad1, Hus1 and Rad17 are essential factors that activate checkpoint signalings. In mammals, there are four mediator-type proteins that contain BRCA1 C-terminus repeat (BRCT) domains that serve protein–phosphoprotein interaction modules. In mammals, signals initiated by the sensors very rapidly transduce to ATM and ATR kinases, which are both extremely large proteins that phosphorylate a great number of substrates. The checkpoint kinases Chk1 and Cds1 (Chk2) were first identified in fission yeast as essential for cell-cycle arrest before mitosis in response to DNA damage or DNA replication blockage, respectively. These kinases were also identified in vertebrate cells based on their homology with yeast scChk1 and scRad53/ spCds1. This protein plays an essential role in DNA damage and the DNA replication checkpoint response. Chk1 is phosphorylated at Ser317/345 in response to DNA damage in both mammals and fission yeast. Chk1 has been shown to phosphorylate Cdc25-A, Cdc25-B and Cdc25-C, which downregulates their phosphatase activity through several distinct mechanisms. In contrast to Chk1, Chk2 is dispensable for pre-natal development. Given that Chk2 is activated by phosphorylation of its threonine residue at 68 in an ATM-dependent manner in response to IR treatment, Chk2 is implicated in the DNA damage signaling pathway. p53 is thought to be essential for G1 arrest in response to DNA damage. The key transcriptional target of p53 is the p21 Cdk inhibitor (p21CKI) which inhibits cyclin E–Cdk2 activity, thereby inhibiting G1/S transition. p21CKI also binds to the cyclin D–Cdk4 complex and prevents it from phosphorylating Rb, thereby suppressing the RB/E2F pathway (Ref.3).

In Schizosaccharomyces pombe or Fission Yeast, the major effector of the replication checkpoint is the protein kinase Cds1. Activation of Cds1 is known to require the upstream kinase Rad3 and the mediator Mrc1, but the biochemical mechanism of activation is not well understood. The replication checkpoint is activated in two stages. In the first stage, Mrc1 recruits Cds1 to stalled replication forks by interactions between the FHA domain of Cds1 and specific phosphorylated Rad3 consensus sites. In the second stage, primed Cds1 molecules dimerize via phospho-specific interactions mediated by the FHA domains and are activated by autophosphorylation (Ref.4).

In the budding yeast Saccharomyces cerevisiae, several checkpoints arrest the cell cycle when DNA damage, replication, or mitotic errors occur. The DNA damage signal is propagated by the kinases Mec1p and Tel1p, a protein functionally redundant with Mec1p. Mec1p and Rad24p (a component of the RFC-like complex which loads the PCNA-like clamp composed of Ddc1p, Rad17p, and Mec3p onto DNA are independently recruited to the site of DNA damage. This localization results in the activation of Mec1p and leads to the activation of Rad53p through the phosphorylation of Rad9p (Ref.5 ).

In Drosophila, ATM checkpoint kinase is minimally involved in controlling the G2/M checkpoint that serves to prevent mitotic entry in the presence of DNA damage. Both ATM and its regulator Mre11 are important for the checkpoint and that their roles become essential when animals are challenged with a low dose of X rays or when they have compromised checkpoint function of the ATM-related ATR kinase. Mei41 and Grp, the ATM/ATR and Chk1 homologs in flies, are involved in the replication-block-induced delay of mitosis in embryonic cells. However, the major role of Grp is to regulate the tyrosine phosphorylation of CDC2 (Ref.6). Emerging data suggest that synthetic lethal interactions between mutated oncogenes or tumor suppressor genes with molecules involved in the DNA damage response and DNA repair pathways can be therapeutically exploited to preferentially kill cancer cells (Ref. 7).