PerspectiveCELL CYCLE

Rad9 Comes of Age

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Science  10 Jul 1998:
Vol. 281, Issue 5374, pp. 185-186
DOI: 10.1126/science.281.5374.185

Normal cells that have sustained damage to their DNA wisely stop dividing, halted at the entry point of mitosis. [HN2], [HN3], [HN4], [HN5], [HN6] But budding yeast (Saccharomyces cerevisiae) cells with mutations in the gene RAD9 are exceedingly imprudent. [HN7], [HN8] They ignore the presence of breaks in the double-stranded DNA of their genome and proceed unchecked into mitosis (1)—a life-threatening situation roughly akin to embarking on a long car journey with holes in the tires. Even before the impetuosity of the rad9 mutants became apparent 10 years ago (1), it was known that eukaryotic cells delayed their entry into mitosis when their DNA was damaged, but the rad9 mutant pointed to a specific extrinsic mechanism for this effect. As the basis of this so-called checkpoint has been genetically dissected, we have learned much about mitosis and DNA repair in eukaryotic cells. [HN9], [HN10], [HN11] We have, however, discovered surprisingly little about the Rad9 protein itself. A report on page 272 of this issue (2) begins to correct that shortcoming.

Proteins with some similarity to Rad9 have been identified in species from human to Schizosaccharomyces pombe. A region of homology at the COOH-terminus of Rad9—the BRCT domain—is shared between Rad9 and at least two other gene products, BRCA1 [HN12]. [HN13] and 53BP1, both of which have intriguing connections to human cancer (3). BRCA1, a putative tumor suppressor, is linked to breast cancer, and 53BP1 is a protein that binds to the tumor suppressor p53 (4), although neither of these proteins are true homologs of budding yeast RAD9. The S. pombe homolog of RAD9, called both rhp9 (5) and crb2 (6), functions in much the same way as the S. cerevisiae Rad9 protein (5, 6): Mutants lacking rhp9/crb2 function fail to arrest in the cell cycle when DNA is damaged or when DNA replication is impaired by inactivating DNA polymerases or DNA ligase.

Sun et al. encountered Rad9 in a search for proteins that could interact with the protein kinase encoded by RAD53. Rad53 is an essential protein kinase in S. cerevisiae [HN14], [HN15] required for DNA replication and for cell cycle arrest in response to replication blocks and DNA damage (7). Rad53 is phosphorylated when DNA is damaged, and this phosphorylation depends on a number of other gene products (8). Rad9 function is necessary for phosphorylation of Rad53 when cells in G1 or G2 are exposed to DNA damage (9). Using a kinase-defective allele of Rad53, Sun et al. performed a two-hybrid protein interaction screen and isolated Rad9 (2). They show that Rad9 is modified in response to DNA damage (probably by phosphorylation) and that the modified form of Rad9 is selectively bound by Rad53. Furthermore, Rad9 interacts with the COOH-terminal domain of Rad53.

Rad53 has two FHA (forkhead-associated) domains, one in the NH2-terminal region (FHA1) and one in the COOH-terminal region (FHA2). The FHA domain, originally described as a region of homology in a subset of the family of forkhead-type transcription factors (10), lies outside of the DNA binding domain conserved in all forkhead transcription factors and consists of a stretch of 55 to 75 amino acids with three highly conserved blocks of residues. This motif is also found in several proteins that are not transcription factors, including Rad53 and its fission yeast homolog, Cds1. Mutations in FHA2 of Rad53 diminish its capacity to associate with Rad9, abolish DNA damage-dependent phosphorylation of Rad53, eliminate G2/M arrest, and increase RNR3 transcription (2). Thus, the ability of Rad53 to associate with phosphorylated Rad9 correlates with its ability to function in the DNA damage response, suggesting that this association is physiologically relevant and that phosphorylation of Rad9 is a necessary component of the response.

There is precedence for the recognition of a phosphorylated partner by an FHA domain-containing protein. The Arabidopsis thaliana protein KAPP (kinase-associated protein phosphatase) binds to the serine-threonine receptor-like protein kinase RLK5 (11). The domain of KAPP that binds to RLK5 (originally called the KI domain for “kinase interacting”) includes an FHA domain (10). Like Rad9, RLK5 must be phosphorylated to bind to this region of its partner (11). Thus, FHA domains may be analogous to SH2 (Src homology 2) domains that recognize tyrosine-phosphorylated residues to mediate signal transduction [HN16] through cell surface receptors. It will be of interest to know whether mutations in the conserved residues of KAPP FHA abolish binding to RLK5, and to confirm that conserved residues within FHA domains of other proteins are important for function and that phosphorylation of the binding partner is necessary for FHA-mediated association.

Mutations in the COOH-terminal FHA2 domain prevent Rad53 from responding to some types of DNA damage—alkylation by methyl methane sulfonate or production of single-stranded DNA by inactivation of cdc13. FHA2 mutations, however, do not prevent Rad53 phosphorylation or cell cycle arrest when DNA replication is inhibited by hydroxyurea (2, 12), nor does it confer sensitivity to ultraviolet (UV) light (12). Therefore, this domain is not solely responsible for Rad53's role in the DNA damage response; the NH2-terminal FHA1 domain may participate as well. Consistent with a role for FHA1, deletion of the NH2-terminal FHA1 domain confers sensitivity both to hydroxyurea and to UV light (12). Perhaps the two domains mediate Rad53 function at different points in the cell cycle—FHA1 during S phase and FHA2 during G1 and G2 (see the figure). This hypothesis is consistent with the observation that Rad53's response to UV light depends on Rad9 during G1 and G2, and on DNA polymerase ε during S phase. A model presents itself: Rad9 binds to FHA2 to mediate the Rad53 response during G1 or G2 while another protein, perhaps polymerase ε binds to FHA1 to mediate the Rad53 response during S phase (see the figure). If this is the case, then the sensitivity of the FHA1 deletion mutant and resistance of the FHA2 deletion mutant to UV light may be explained by the fact that cells in S phase are more sensitive to UV light. This analysis is complicated by the fact that the FHA1 deletion mutant also reduces the catalytic activity of Rad53 (12). Point mutations in the FHA1 domain (that do not affect catalytic activity) will determine whether FHA1 specifically confers sensitivity to UV light.

Two ways to stop.

In G1/G2 phase of the cell cycle, DNA damage triggers arrest of the cell cycle via Rad9's interaction with one FHA domain of the kinase RAD53. In S phase the same arrest occurs via interaction of an unknown, protein (x) with Rad53's other FHA domain.

The gene cds1 encodes a homolog of Rad53 in fission yeast (13), but its function is not entirely parallel. Cds1 has only a single FHA domain, and mutants lacking cds1 function have only some of the phenotypes of rad53 mutants in budding yeast. Like rad53 mutants, cells without cds1 function lose viability when exposed to either replication blocks or DNA damage; unlike rad53 mutants, however, they arrest the cell cycle in either case (14). Thus, Cds1 does not share Rad53's checkpoint function. Even so, both proteins clearly respond to DNA damage in a cell cycle-specific fashion. Cds1 activity is increased by DNA damage, but only during S phase (14). Rad53 requires Rad9 to function during G1 and G2, but not during S phase. The FHA domain in Cds1 may be analogous to the FHA1 domain of Rad53 in conferring S phase-specific regulation on these kinases. The second function of Rad53, to mediate arrest in response to DNA damage, is provided by the fission yeast protein kinase Chk1 (15). The fission yeast homolog of Rad9, Crb2, binds Chk1 (6). Although Crb2 is phosphorylated in response to DNA damage, it is not yet known whether phosphorylation of Crb2 or its association with Chk1 is necessary for Crb2 function in fission yeast. Chk1 does not have an obvious FHA domain, suggesting that the Crb2-Chk1 interaction may be mediated by another mechanism.

Rad9 joins a growing list of proteins implicated in the cell cycle checkpoint pathway that are phosphorylated in response to DNA damage. Which kinases are responsible for these events? Certainly a number of protein kinases function along the checkpoint pathways, but we are still a long way from understanding how they are regulated by DNA damage or replication blocks and what their in vivo substrates actually are. The protein kinases thus far implicated in regulating the damage response in S. cerevisiae—Mec1, Tel1, and Rad53—are thought to function downstream of Rad9. The results of Sun et al., however, inform us that Rad9 phosphorylation may in fact be dependent on these kinases (2), implying that they act upstream of Rad9. Either way, an additional as yet unidentified kinase could be involved, and the pathways are more complicated than we have thought.

How best to dissect this complex pathway of interacting proteins, kinases, and substrates? Forge ahead with open minds. A combination of genetics, cell biology, and biochemistry has gotten us into this tangle. Let us hope that these approaches can eventually lead us out.

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.

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.

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.

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.

DNA Repair is one of the topics covered by the Laboratory of Molecular Genetics.

Numbered Hypernotes

1. Nancy C. Walworth's Web page describes her research and lists recent publications.

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

3. Mitosis provides illustrations and movies of the cell cycle.

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

5. Virtual Mitosis, developed by the Department of Biological Sciences at the University of Cincinnati, presents photomicrographs of the mitotic process and defines terms.

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

7. Molecular Genetics by Ulrich Melcher is a tutorial designed to accompany a course for beginning graduate students taught at Oklahoma State University. Replication and Mitosis includes a discussion of RAD9.

8. RAD9 is described in the Yeast Protein Database. The Saccharomyces Genome Databank (SGD) provides additional information about RAD9.

9. DNA Replication is a basic introduction to DNA synthesis and repair.

10. DNA Repair in Three Dimensions is a review of DNA repair mechanisms that was published in Trends in Biochemical Sciences in 1996.

11. DNA Replication is one chapter of a Web tutorial developed by Phillip E. McClean for a course in Intermediate Genetics. This page describes the proteins involved in DNA replication and provides links to related chapters of the tutorial and to other Web sites.

12. Breast Cancer and the BRCA1 Gene: Questions and Answers is a page produced by the National Cancer Institute that describes BRCA1.

13. The Breast Cancer and BRCA1 2 3 Information Directory is a collection of resources for understanding the link between BRCA1 and breast cancer.

14. Classification of Saccharomyces cerevisiae Protein Kinases includes a classified list with short descriptions of the protein kinases of S. cerevisiae.

15. 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.

16. Signal Transduction is a module of the THCME Medical Biochemistry course developed by the Indiana University School of Medicine. This module covers the classification of cell surface receptors and the role of tyrosine phosphorylation in signal transduction.

17. Department of Pharmacology, Robert Wood Johnson Medical School, University of Medicine and Dentistry of New Jersey.

References

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