PerspectiveCELL CYCLE

A DNA Damage Checkpoint Meets the Cell Cycle Engine

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Science  05 Sep 1997:
Vol. 277, Issue 5331, pp. 1450-1451
DOI: 10.1126/science.277.5331.1450

Cell cycle regulators [HN2] govern key transitions in the life of a cell—when to begin DNA replication and when to enter mitosis and divide. Preeminent among cell cycle regulators is the family of cyclin-dependent kinases (p34CDK) and their partner cyclins, [HN3] which together form heterodimer protein kinases (1). Reflecting that preeminence, Cdk-cyclin was crowned the cell cycle's “engine” (2): How goes Cdk-cyclin, so goes the cell cycle.

Changes in cell physiology, particularly damage to DNA, stop the cell cycle either before DNA replication in G1 (termed the G1 checkpoint) or before mitosis in G2 [the G2-M checkpoint (3, 4)]. In many cell types DNA damage response pathways cause arrest by regulation of Cdk-cyclin through checkpoint proteins, which sense damage and transduce an inhibitory signal (3). Until recently it was unclear whether Cdc2, [HN4] a prominent member of this Cdk family and a major mitotic activator in yeast, even plays a role in arrest in G2 after damage. Nor was it clear how the checkpoint proteins transmit a signal to cause arrest. In the last year, the functions of both Cdc2 and checkpoint proteins have become clearer, and an ever more detailed hypothesis for a checkpoint pathway has emerged, culminating in three reports (pages 1495, 1497, and 15011501) in this week's issue from fission yeast, human, and mouse (57).

Evidence from several cell types has indicated that the G2 arrest is caused by regulation of Cdc2; DNA damage results in the phosphorylation of inhibitory sites on the Cdc2 catalytic subunit in the filamentous fungi Aspergillus, in human cells, and in fission yeast (810). This inhibitory phosphorylation is required for arrest at the G2-M checkpoint: in all three organisms nonphosphorylatable mutants of Cdc2 fail to fully arrest. How is this inhibitory phosphorylation of Cdc2 achieved? This question is addressed in three reports in this issue, which forge an attractive model explaining how upstream checkpoint proteins mediate the inactivation of Cdc25, a key activator of Cdc2.

The elegant model (see the figure) derived from these three reports seems destined for textbooks—regardless of whether it ultimately proves correct in detail. As yet, the single proposed pathway has not been demonstrated in its entirety in any one cell type but is instead synthesized from experiments in evolutionarily distant organisms—human, mouse, and yeast. Nevertheless, the mosaic nature of this model seems justified because the pathways in different cell types thus far have been found to be conserved.

A DNA break

(a single-strand gap highlighted in yellow) activates the protein kinase Rad3 in fission yeast (and probably Rad3-like proteins ATM and ATR in human cells) (3). Activation of Rad3 probably occurs through association with other checkpoint proteins not shown (3). Active Rad3 then somehow activates the protein kinase Chk1 (Rad3 is required for phosphorylation of Chk1, but the exact mechanism of activation is unknown) (13). Activated Chk1 phosphorylates the phosphatase Cdc25 on Ser216 that then binds to and is sequestered by 14-3-3 protein. Sequestered Cdc25 is prevented from activating Cdc2. Cells arrest in G2 when inhibitory phosphorylation of Cdc2 is intact. The dotted line highlights the aspects of this pathway discussed in three reports in this issue (57).

After DNA damage, the inhibitory phosphorylation of Cdc2 that causes G2 arrest occurs on Tyr15. Genetic studies in fission yeast imply that this phosphorylation is maintained by the activity of protein kinases spWee1 [and the redundant spMik1 (here the prefix sp refers to Schizosaccharomyces pombe] and the simultaneous inactivity of phosphatase spCdc25 (10). In one of the new reports, human Cdc25 (here designated hCdc25) is shown to become phosphorylated in vivo on Ser216 after DNA damage in mammalian cells (7), and all three reports suggest that this is how Cdc25 (the S. pombe or human protein) is kept inactivated (57, 10). This phosphorylation is clearly functionally important because the nonphosphorylatable allele hCdc25(S216A) (in which Ser216 is mutated to Ala) is defective for G2-M arrest.

Phosphorylation of spCdc25 and hCdc25 is achieved by Chk1 protein kinase (57), a protein kinase required for arrest after DNA damage at least in fission yeast (1113). From elegant genetic studies in fission yeast, Furnari et al. argue that Chk1 acts primarily through Cdc25 and not through Wee1 and Mik1 protein kinases, although they are required to maintain arrest (6, 10). [The relevance of the association of spChk1 with spWee1 is as yet unknown (14)]. Chk1 probably acts directly on Cdc25: spChk1 and hChk1 proteins (57) can bind to and phosphorylate spCdc25 and hCdc25 in vivo and in vitro. The sites of phosphorylation on hCdc25 by hChk1 is the same Ser216 (5, 7) that is required for arrest in vivo (7).

How does phosphorylation inhibit Cdc25? Cdc25 phosphorylation itself does not appear to directly inactivate Cdc25 (7). Rather, Peng et al. found that only the species of Cdc25 phosphorylated on Ser216 (and not the unphosphorylated species) binds to 14-3-3 proteins, a class of molecules that bind to signaling molecules including phosphatases (15). Peng et al., therefore, propose that the 14-3-3 protein sequesters Ser216-phosphorylated Cdc25, thereby preventing it from activating Cdc2 by dephosphorylation. Supporting this scheme is the observation that 14-3-3 proteins encoded by the rad24 and rad25 genes are involved in arrest after damage in fission yeast (16, 17).

Taken together, these results suggest that after damage, Chk1 becomes active, phosphorylates Cdc25 on Ser216, which promotes the binding of Cdc25 to 14-3-3 protein and therefore its sequestration. In this state, Cdc25 cannot activate Cdc2. The activation of Chk1 after DNA damage appears to involve phosphorylation mediated by other checkpoint proteins, including spRad3 in fission yeast (13) and probably related Rad3-like proteins in other cell types (3).

This hypothesis now connects the function of checkpoint protein kinase Chk1 to cell cycle regulators Cdc25 and Cdc2. Because this model is based on a number of inferences and depends on the conservation of pathways in different cell types, several points remain to be directly tested. The roles of the putative Chk1 and 14-3-3 proteins in mammalian cells in arrest have yet to be established. The model predicts that the fission yeast Rad24 and Rad25 proteins will bind to and sequester Cdc25 phosphorylated on Ser216 and thereby somehow prevent activation of Cdc2. If and how that occurs is unknown. The mechanism of activation of Chk1 remains to be determined as well.

These findings suggesting a specific mechanism for cell cycle arrest at G2 (see the figure) are a substantial advance in the field. Nevertheless, additional mechanisms of regulation for G2 arrest apparently exist. Human cells with a nonphosphorylatable Cdc2 subunit are only partially defective in G2 arrest, leading to speculation that other mechanisms (such as localization of cyclin B) may also be involved in arrest (9). In budding yeast, inhibitory phosphorylation of Cdc28, the homolog of Cdc2, is not sufficient to explain G2 arrest (20, 21). Finally, arrest in S phase when DNA replication is blocked may in some cell types also require Cdc2 phosphorylation (7), but in some cases S-phase arrest involves additional mechanisms unrelated to Cdc2 phosphorylation (1820). Indeed, arrest in G1 after DNA damage in mammalian cells may be regulated by both an inhibitor of Cdk activity, p21 (23, 24), and an inhibitory tyrosine phosphorylation of Cdk4 (25) (possibly by the same Chk1-dependent pathway discussed here).

Next, the field will want to inactivate the checkpoint pathways for cancer therapy by targeting components like Chk1 (26). Cancer cells treated with drugs that inactivate Chk1, for example, may render those cells more sensitive to DNA damaging agents already widely used in therapy. Whether the resulting increase in sensitivity will affect cancer cells more than normal cells in one of several important issues. Ultimately, to manipulate checkpoint pathways we will need to know yet more molecular details of those pathways, how the pathways are changed in cancer cells, and what else the specific proteins in those pathways do in a cell.

HyperNotes Related Resources on the World Wide Web

General Hypernotes

The Dictionary of Cell Biology (London, Academic Press, 1995) defines some of the terms used in this article.

Cell Division and the Cell Cycle, developed at the University of Alberta, describes the cell cycle and includes a glossary of terms.

Mitosis provides illustrations and movies of the cell cycle.

Mitosis, developed at McGill University, provides high-quality animations of mitosis and cell division. DNA Replication illustrates the biosynthesis of DNA.

Virtual Mitosis, developed by the Department of Biological Sciences at the University of Cincinnati, presents photomicrographs of the mitotic process and defines terms. It also offers a link to Virtual Meiosis, in which the phases of meiosis are illustrated.

Cell Cycle is a page in the Cytogenetic Terms Index. Cell cycle is defined with links to descriptions of cell division and the phases of mitosis.

Diagrams of the phases of meiosis and mitosis are offered in the About Biotech Graphics Gallery, a component of Access Excellence, a national educational program sponsored by Genentech, Inc.

The MIT Biology Hypertextbook, developed by the Experimental Study Group at the Massachusetts Institute of Technology, provides background information on the biology of cells and nucleic acids. Mitosis, a section within Cell Biology, provides descriptions, and diagrams of the phases of the cell cycle.

The Protein Kinase Resource (PKR) is a web accessible compendium of information on the protein kinase family of enzymes. This resource includes tools for structural and computational analyses as well as links to related information maintained by others.

Pedro's BioMolecular Research Tools is a collection of WWW links to information and services useful to molecular biologists. It provides links to molecular biology search and analysis tools; bibliographic, text, and Web search services; guides and tutorials; and biological and biochemical journals and newsletters.

The World Wide Web Virtual Library: Biosciences points to virtual library pages for Biological Molecules, and Biochemistry and Molecular Biology. Each of these pages presents a long list of Web resources. The World Wide Web Virtual Library Biological Molecules covers molecular sequence and structure databases, metabolic pathway databases, and other lists of Web resources. The World Wide Web Virtual Library: Biochemistry and Molecular Biology is a list of resources listed by provider.

Cell & Molecular Biology Online is a well-organized list of Web resources for cell and molecular biologists. For each resource, a brief description is provided.

CSUBIOWEB, the California State University Biological Sciences Web server, provides links to other Web sites on cell biology and molecular biology.

Numbered Hypernotes

1. Ted Weinert describes his research and lists some of his publications.

2. An On-Line Biology Book by M. J. Farabee includes a chapter on Cell Division: Binary Fission and Mitosis. This chapter describes the phases of the cell cycle and provides background information on cell cycle regulation. Several diagrams are included and links to related Web resources are available.

3. Cell Cycle Genes is a chapter of the Interactive Fly, an Internet guide to Drosophila genes and their role in development. The cell cycle in Drosophila is described and the role of the cyclins in regulating the cell cycle is outlined. A biological overview and a discussion of evolutionary homologs refer to cell cycle regulation in other organisms.

4. CDC2, CDC25A, CDC25B, and CDK2 are described in Online Mendelian Inheritance in Man (OMIM), a catalog, edited by Victor A. McKusick, of human genes and genetic disorders. For each gene, OMIM provides general information on the function of the gene, references derived from MEDLINE, and links to gene maps and molecular sequence data.

5. Department of Molecular and Cellular Biology, the University of Arizona.


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