Cell cycle checkpoint signaling through the ATM and ATR kinases

The genomes of eukaryotic cells are under continuous assault by environmental agents (e.g., UV light and reactive chemicals) as well as the byproducts of normal intracellular metabolism (e.g., reactive oxygen intermediates and inaccurately replicated DNA). Whatever the origin, genetic damage threatens cell survival, and, in metazoans, leads to organ failure, immunodeficiency, cancer, and other pathologic sequelae. To ensure that cells pass accurate copies of their genomes on to the next generation, evolution has overlaid the core cell-cycle machinery with a series of surveillance pathways termed cell-cycle checkpoints. The overall function of these checkpoints is to detect damaged or abnormally structured DNA, and to coordinate cell-cycle progression with DNA repair. Typically, cell-cycle checkpoint activation slows or arrests cell-cycle progression, thereby allowing time for appropriate repair mechanisms to correct genetic lesions before they are passed on to the next generation of daughter cells. In certain cell types, such as thymocytes, checkpoint proteins link DNA strand breaks to apoptotic cell death via induction of p53. Hence, loss of either of two biochemically connected checkpoint kinases, ATM or Chk2, paradoxically increases the resistance of immature (CD4CD8) T cells to ionizing radiation (IR)-induced apoptosis (Xu and Baltimore 1996; Hirao et al. 2000). In a broader context, cell-cycle checkpoints can be envisioned as signal transduction pathways that link the pace of key cell-cycle phase transitions to the timely and accurate completion of prior, contingent events. It is important to recognize that checkpoint surveillance functions are not confined solely to the happenings within the nucleus–extranuclear parameters, such as growth factor availability and cell mass accumulation, also govern the pace of the cell cycle (Stocker and Hafen 2000). However, for the purposes of this review we will focus exclusively on the subset of checkpoints that monitor the status and structure of chromosomal DNA during cell-cycle progression (Fig. 1). These checkpoints contain, as their most proximal signaling elements, sensor proteins that scan chromatin for partially replicated DNA, DNA strand breaks, or other abnormalities, and translate these DNA-derived stimuli into biochemical signals that modulate the functions of specific downstream target proteins. Despite the recent explosion of information regarding the molecular components of cell-cycle checkpoints in eukaryotic cells, we still have only a skeletal understanding of both the identities of the DNA damage sensors and the mechanisms through which they initiate and terminate the activation of checkpoints. However, members of the Rad group of checkpoint proteins, which include Rad17, Rad1, Rad9, Rad26, and Hus1 (nomenclature based on the Schizosaccharomyces pombe gene products) are widely expressed in all eukaryotic cells, and are prime suspects in the lineup of candidate DNA damage sensors (Green et al. 2000; O’Connell et al. 2000). Three of these Rad proteins, Rad1, Rad9, and Hus1, exhibit structural similarity to the proliferating cell nuclear antigen (PCNA), and accumulating evidence supports the idea that this similarity may extend to function as well (Thelen et al. 1999; Burtelow et al. 2000). During DNA replication, PCNA forms a homotrimeric complex that encircles DNA, creating a “sliding clamp” that tethers DNA polymerase to the DNA strand. Rad1, Rad9, and Hus1 are also found as a heterotrimeric complex in intact cells, and it has been postulated that the Rad1–Rad9–Hus1 complex encircles DNA at or near sites of damage to form a checkpoint sliding clamp (CSC) (O’Connell et al. 2000), which could serve as a nucleus for the recruitment of the checkpoint signaling machinery to broken or abnormally structured DNA. The analogy between PCNA and the Rad1–Rad9–Hus1 complex extends even further. The loading of the PCNA clamp onto DNA is controlled by the clamp loading complex, replication factor C (RFC). Interestingly, yet another member of the Rad family, Rad17, bears homology to the RFC subunits and, in fact, associates with RFC subunits to form a putative checkpoint clamp loading complex (CLC) that governs the interaction of the Rad1– Rad9–Hus1 CSC with damaged DNA (Green et al. 2000; O’Connell et al. 2000). Although this model is fascinating, rigorous biochemical evaluations of the interplay between the CLC and CSC complexes, and the interactions of both complexes with damaged chromatin, are needed before the model can be accepted without reservation. Present address: The Burnham Institute, 10901 N. Torrey Pines Road, La Jolla, CA 92037, USA E-MAIL abraham@burnham.org; FAX (858) 713-6268. Article and publication are at http://www.genesdev.org/cgi/doi/10.1101/ gad.914401.