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00.353.91.512504; Email: noel.lowndes@nuigalway.ie. Received 11/12/03; Accepted 11/17/03. Previously published online as a Cell Cycle E-publication:.
[Cell Cycle 3:2, 119-122; February 2004]; ©2004 Landes Bioscience

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Remodelling the Rad9 Checkpoint Complex Preparing Rad53 for Action ABSTRACT

*Correspondence to: Noel F. Lowndes; Genome Stability Laboratory; Department of Biochemistry and National Centre for Biomedical Engineering Science; National University of Ireland Galway; Galway, Ireland; Tel.: 00.353.91.750309; Fax: 00.353.91.512504; Email: [email protected]

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Received 11/12/03; Accepted 11/17/03

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Genome Stability Laboratory; Department of Biochemistry and National Centre for Biomedical Engineering Science; National University of Ireland Galway; Galway, Ireland

DNA damage checkpoints are signal transduction pathways that are activated after genotoxic insults to protect genomic integrity. The Rad9 protein functions in the DNA damage checkpoint pathway in Saccharomyces cerevisiae and is essential for the Mec1-dependent activation of the effector kinase Rad53. We recently described the purification of two soluble distinct Rad9 complexes. The large 850 kDa complex consists of hypophosphorylated Rad9 and the chaperone proteins Ssa1/2. This complex is found both in undamaged cells as well as in cells treated with DNA damaging agents. The smaller 560 kDa complex contains hyperphosphorylated Rad9, Ssa1/2 and, in addition, Rad53. This complex forms only in cells with compromised DNA integrity. Once bound to the smaller complex, Rad53 can be activated by in trans autophosphorylation. Here, we propose a model in which the large Rad9 complex is remodelled after a genomic insult by chaperone activity to a smaller Rad53 activating complex.

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Michael van den Bosch Noel F. Lowndes*

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Previously published online as a Cell Cycle E-publication: http://www.landesbioscience.com/journals/cc/abstract.php?id=652

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The authors thank members of the Lowndes’ laboratory for their comments on the manuscript. MvdB is sponsored by the Health Research Board (HRB) Ireland.

The genetic information of eukaryotic organisms is encrypted in the two complementary strands of the DNA. However, the structure and integrity of this blueprint for proper development, reproduction and cell functioning is constantly being threatened by numerous exogenous and endogenous sources. The ability of cells to deal with spontaneous and/or environmentally induced DNA damage is critical to both genome stability and cell viability. Consequently, cells have evolved surveillance mechanisms, collectively termed DNA damage checkpoints that monitor genome integrity and, in the presence of damage, regulate appropriate cellular responses. Once an abnormal DNA structure is detected, the DNA damage checkpoint activates pathways that transiently halt cell cycle progression (typically in the G1, or G2/M phases) or slow down the rate at which DNA synthesis (S-phase) proceeds. In addition to delaying cell cycle progression, the DNA damage checkpoint controls the transcriptional induction of a large regulon, including genes with roles in most DNA repair pathways. In the presence of persistent or irreparable DNA lesions, yeast cells eventually downregulate the checkpoint signal and attempt to resume normal cell cycling, whereas in higher eukaryotes an apoptotic program is activated which effectively removes the damaged cells from the organism. At present, DNA damage checkpoint pathways are best understood in yeast model systems. In the budding yeast, Saccharomyces cerevisiae, RAD9 is considered the prototypical DNA damage checkpoint gene. The Rad9 protein is required for full DNA damage checkpoint pathway activity throughout the cell cycle, although it is not required for the checkpoint triggered by nucleotide depletion after treatment of cells with hydroxyurea1-3 Rad9 is a phosphoprotein that is hypophosphorylated during normal cell cycle progression and hyperphosphorylated after treatment of cells with a wide range of DNA damaging agents.4,5 The identity of the kinase responsible for hypophosphorylation has yet to be reported but hyperphosphorylation after DNA damage is principally dependent on Mec1 (the related Tel1 kinase plays a role only when cells are growing sub-optimally).4 Both Mec1 and Tel1 are protein kinases belonging to the family of phosphatidylinositol-(3’) kinase-like protein kinases (PIKKs) that includes Schizosaccharomyces pombe Rad3 and ATM and ATR in mammals.

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ACKNOWLEDGEMENTS

INTRODUCTION

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DNA damage checkpoint, Mec1, Rad9, Rad53, Chaperones, Ssa1/2

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KEY WORDS

CHAPERONE PROTEINS ARE REQUIRED FOR RAD9 FUNCTION We recently characterised two distinct soluble Rad9 complexes in cells with compromised DNA integrity—a large (>850 kDa) complex containing cell-cycle modified www.landesbioscience.com

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translocating proteins through organellar membranes, facilitating the degradation of unstable proteins and controlling the biological activity of folded regulatory proteins.8 It is tempting to speculate, but remains to be proven, that the Ssa1/2 proteins are required for remodelling the large 850 kDa hypophosphorylated Rad9 complex after DNA damage into the smaller 560 kDa complex and release of Rad53 from the smaller complex.

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(hypophosphorylated) Rad9 and a smaller (560 kDa) complex containing hyperphosphorylated Rad9 as well as the effector kinase Rad53.6 In undamaged cells only the hypophosphorylated 850 kDa complex can be detected. Thus following DNA damage, Rad9 is hyperphosphorylated by Mec1 which somehow triggers remodelling of the large Rad9 complex and results in the formation of the smaller 560 kDa complex that associates with Rad53.6 We have purified both the large, as well as the small complex and determined that both Rad9 complexes also contain the highly homologous 70 kDa chaperone proteins, Ssa1 and/or Ssa2.7 Consistent with this observation, genetic experiments indicate a redundant role for the SSA1 and SSA2 genes in survival, G2/M checkpoint regulation and phosphorylation of both Rad9 and Rad53 after UV irradiation. Ssa1 and Ssa2 belong to a family of four (Ssa1-Ssa4) highly related molecular chaperone proteins of the heat-shock protein 70 (Hsp70) family.8 Ssa1 and Ssa2 are 97% identical and are constitutively expressed to high levels (estimates range from 60,000 to over 250,000 molecules of each per cell compared to our estimates of just 500 molecules of Rad9 per cell), whereas Ssa3 and Ssa4 have ~80% identity to Ssa1/2 and are reported to be 15-fold less expressed in exponentially growing cells.7,9,10 However, after heat shock both Ssa3 and Ssa4 are induced to high levels. Moreover, in ssa1∆ssa2∆ cells SSA3 and SSA4 are constitutively expressed but are unable to fully compensate for the lack of Ssa1 and Ssa2. ssa1∆ssa2∆ cells are characterised by slow growth and temperature sensitivity and mutation of all four SSA genes results in lethality.11,12 In order to verify whether Ssa3 and Ssa4 can also be recruited to the Rad9 complex we purified the large Rad9 complex from exponentially growing ssa1∆ssa2∆ cells, in which the normally inducible SSA3 and SSA4 genes are now constitutively expressed. We found that Ssa3 and Ssa4 can indeed be recruited to the Rad9 complex in the absence of Ssa1 and Ssa2.7 Thus, although Ssa3 and Ssa4 can bind the Rad9 complex in ssa1∆ssa2∆ cells, they cannot fully substitute for Ssa1/2 in this complex. Furthermore, we observed that less Rad9 is present in ssa1∆ssa2∆ cells, a phenomenon that might be related to the stabilising effect that chaperones have on protein complexes. Other roles for Hsp70 chaperones include disassembling oligomeric protein structures, folding of some newly translated proteins, guiding

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Figure 1. Remodelling of the Rad9 complex and activation of the protein kinase Rad53. After DNA damage, remodelling of chromatin surrounding the damage site and phosphorylation of histone H2A occurs. A role for Rad9 in sensing DNA damage can’t be excluded but for clarity has not been shown here. The large hypophosphorylated Rad9 complex diffuses to the lesion site or is perhaps actively recruited to the site of DNA damage and is phosphorylated by Mec1. Hyperphosphorylated Rad9, that now provides docking sites for Rad53, is released into the nucleoplasm. Rad53 is transiently recruited to the lesion site and phosphorylated by Mec1 and now able to bind the hyperphosphorylated Rad9 complex. Hyperphosphorylated Rad9 provides a matrix for Rad53 in trans autophosphorylation. During checkpoint recovery hyperphosphorylated Rad53 is released, no further Mec1-dependent phosphorylation of Rad53 occurs and Rad9 probably aggregates into its larger hypophosphorylated form. The chaperone proteins Ssa1/2 are thought to be important for remodelling of the Rad9 complex. Mec1-dependent phosphate residues are shown as blue spheres. Rad53-dependent phosphate residues are shown in green.

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ACTIVATION OF THE CHECKPOINT KINASE RAD53 Recent observations from our group and others suggest that hyperphosphorylation of Rad9 and the smaller Rad9 complex are required for the activation of the checkpoint kinase Rad53.6,7,13 Although many of the molecular details are not known or remain to be fully elucidated, the model in Figure 1 illustrates our view of the events that precede the hyperphosphorylation and, thus, activation of Rad53. In mammalian cells, ATR has been proposed to play an important role in sensing DNA damage and is recruited close to the lesion site.14 Mec1 in budding yeast might have similar characteristics. Mec1 that has been recruited to the vicinity of a DNA lesion can phosphorylate histone H2A.15,16 The extent of this phosphorylation is likely to extend over several kilobases either side of the lesion. In addition, it is likely that the Rad53 protein kinase is also recruited to the vicinity of the DNA lesion where Mec1 can phosphorylate it. The faint Rad53-GFP signal, relative to other checkpoint proteins, observed by Melo et al.17 in nuclear structures termed ‘foci’ indicates that Rad53 has a weaker affinity for foci or is only transiently recruited to these structures. Foci are microscopically visible structures that form in the nucleus of cells after DNA damaging treatments and are thought to correspond to sites of DNA repair and DNA damage-dependent signalling.18 Given that the targets of the Rad53 protein kinase are distributed throughout the nucleoplasm it is likely that after phosphorylation by Mec1, Rad53 is quickly released from the foci into the nucleoplasm. In agreement with this hypothesis, recent observations in human cells by Lukas et al.19 have demonstrated that the mammalian orthologue of Rad53, Chk2, continued to move rapidly throughout the entire nucleus, irrespective of DNA damage.

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and Rad53 synthesis may be required to restore Rad53 to its hypophosphorylated state.

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In summary, emerging evidence suggests that remodelling of checkpoint complexes, both in the vicinity of the lesion and in the nucleoplasm, is an important aspect of checkpoint regulation. The identification of specific chaperone proteins as components of two soluble (nucleoplasmic) Rad9 checkpoint complexes that are known to be subject to phosphorylation-dependent modification is intriguing. Chaperone activity may facilitate:

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1. interaction of the hypophosphorylated Rad9 complex, either directly or indirectly, with the lesion (a role for Rad9 in sensing has not been ruled out); 2. Mec1-dependent phosphorylation and remodelling into the smaller hyperphosphorylated Rad9 complex; 3. release of hyperphosphorylated Rad9 from the vicinity of the lesion; 4. interaction of Mec1 phosphorylated (not hyperphosphorylated) Rad53 with docking sites on hyperphosphorylated Rad9; 5. catalysis of Rad53 in trans autophosphorylation; 6. release of fully active (hyperphosphorylated) Rad53 from the Rad9 complex; 7. reassembly of the hypophosphoylated Rad9 complex after recoverydependent removal of Mec1-dependent phosphate residues.

Given the complexity of the focal structures that assemble around damaged DNA and the dynamic nature of the repair and signalling molecules that are recruited and released continuously from these structures, we are of the opinion that chaperone activities will be required to facilitate many of the multiple protein transitions required to effect a proper cellular response to DNA damage.

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As discussed above, Mec1 is also required for the hyperphosphorylation of Rad9 and given the focal localisation of Mec1 this phosphorylation event is likely to happen close to the DNA within the structure of a focus. Thus the hypophosphorylated Rad9 complex has to diffuse to, or is perhaps actively recruited to, the Mec1 kinase. Similarly to Rad53-GFP, only extremely faint Rad9GFP foci in a background of more uniform nuclear staining have been observed in yeast cells treated with genotoxic agents, suggesting that the majority of Rad9 is retained in the nucleoplasm (Ian Dobbie, unpublished data).17 Hyperphosphorylation of Rad9 by Mec1 results in transition from the large Rad9 complex to the smaller complex. Although it remains to be determined, we consider it highly likely that the chaperone proteins Ssa1 and Ssa2 facilitate the Mec1dependent remodelling of Rad9 complexes. The smaller complex containing hyperphosphorylated Rad9 now provides docking sites for Rad53, which has itself been previously phosphorylated by Mec1. The phosphorylation of Rad53 by Mec1, which does not produce a detectable mobility shift in SDS-PAGE, might be required to ‘prime’ Rad53 for its subsequent docking onto Rad9. Rad53 contains two phospho-residue binding FHA (Forkhead-associated) domains, both of which contribute to the interaction with the hyperphosphorylated form of Rad9.20 It has been shown that the damage-induced Rad9 phosphorylation sites are required for the direct interaction of hyperphosphorylated Rad9 with Rad53.21 While bound to the smaller Rad9 complex, we have proposed that Rad53 molecules are brought into close proximity and become hyperphosphorylated by in trans autophosphorylation.6 In effect, hyperphosphorylated Rad9 acts analogously to a solid state catalyst, where reactants are adsorbed in close proximity onto a surface thereby facilitating their interaction and release of the product which has lower affinity for that surface. Hyperphosphorylated Rad53 molecules must be released from the Rad9 complex in order to target their substrates, a process that may also require the chaperone activity of the Ssa1/2 proteins in the hyperphosphorylated Rad9 complex. Evidence in support of this hypothesis comes from our observation that the release of hyperphosphorylated Rad53 from the smaller Rad9 complex is dependent on ATP and stimulated by potassium ions.6 Interestingly, Ssa1 and Ssa2 are potassium dependent ATP-ases.22 Thus biochemical data, together with our genetic observations that Ssa1/2 are required for Rad9 function, support a role for these chaperones in mediating Rad53 activation. Once released and fully activated, hyperphosphorylated (i.e., by in trans autophosphorylation) Rad53 kinase targets components of the cell cycle machinery to inhibit cell cycle progression, activate DNA repair and target transcription factors to effect the transcriptional response to DNA damage.23 Effective cellular responses to DNA damage result in the full repair of lesions and the resumption of cellular proliferation. We presume that exit from this response requires dephosphorylation of Mec1-dependent phosphorylation sites together with further remodelling of the hyperphosphorylated 560 kDa Rad9 complex, so that the larger hypophosphorylated Rad9 complex, characteristic of undamaged cells, can reform. Indeed, the extent of Rad9 hyperphosphorylation correlates precisely with the checkpoint signal, peaking when the maximal amount of cells are arrested and declining as cells exit the checkpoint.4,24 This suggests active removal of phosphate residues by an unidentified protein phosphatase specific to sites of Mec1-dependent phosphorylation. In contrast, Rad53 hyperphosphorylation, that ocurred after in trans autophosphorylation (which is independent from Mec1 phosphorylation), persists even as cells resume proliferation, suggesting that protein turnover www.landesbioscience.com

References 1. Weinert TA, Hartwell LH. The RAD9 gene controls the cell cycle response to DNA damage in Saccharomyces cerevisiae. Science 1988; 241:317-22. 2. Siede W, Friedberg AS, Friedberg EC. RAD9-dependent G1 arrest defines a second checkpoint for damaged DNA in the cell cycle of Saccharomyces cerevisiae. Proc Natl Acad Sci USA 1993; 90:7985-9. 3. Paulovich AG, Margulies RU, Garvik BM, Hartwell LH. RAD9, RAD17, and RAD24 are required for S phase regulation in Saccharomyces cerevisiae in response to DNA damage. Genetics 1997; 145:45-62. 4. Vialard JE, Gilbert CS, Green CM, Lowndes NF. The budding yeast Rad9 checkpoint protein is subjected to Mec1/Tel1-dependent hyperphosphorylation and interacts with Rad53 after DNA damage. EMBO J 1998; 17:5679-88. 5. Emili A. MEC1-dependent phosphorylation of Rad9p in response to DNA damage. Mol Cell 1998; 2:183-9. 6. Gilbert CS, Green CM, Lowndes NF. Budding yeast Rad9 is an ATP-dependent Rad53 activating machine. Mol Cell 2001; 8:129-36. 7. Gilbert CS, van den Bosch M, Green CM, Vialard JE, Grenon M, Erdjument-Bromage H, Tempst P, Lowndes NF. The budding yeast Rad9 checkpoint complex: chaperone proteins are required for its function. EMBO Rep 2003; 4:953-8. 8. Bukau B, Horwich AL. The Hsp70 and Hsp60 chaperone machines. Cell 1998; 92: 351-66. 9. Norbeck J, Blomberg A. Two-dimensional electrophoretic separation of yeast proteins using a non-linear wide range (pH 3-10) immobilized pH gradient in the first dimension; reproducibility and evidence for isoelectric focusing of alkaline (pI > 7) proteins. Yeast 1997; 13:1519-34. 10. Ghaemmaghami S, Huh WK, Bower K, Howson RW, Belle A, Dephoure N, O’Shea EK, Weissman JS. Global analysis of protein expression in yeast. Nature 2003; 425:737-41. 11. Craig EA, Jacobsen K. Mutations of the heat inducible 70 kilodalton genes of yeast confer temperature sensitive growth. Cell 1984; 38:841-9. 12. Baxter BK, Craig EA. Suppression of an Hsp70 mutant phenotype in Saccharomyces cerevisiae through loss of function of the chromatin component Sin1p/Spt2p. J Bacteriol 1998; 180:6484-92. 13. Sun Z, Hsiao J, Fay DS, Stern DF. Rad53 FHA domain associated with phosphorylated Rad9 in the DNA damage checkpoint. Science 1998; 281:272-4. 14. Zou L, Elledge SJ. Sensing DNA damage through ATRIP recognition of RPA-ssDNA complexes. Science 2003; 300:1542-8. 15. Downs JA, Lowndes NF, Jackson SP. A role for Saccharomyces cerevisiae histone H2A in DNA repair. Nature 2000; 408:1001-4.

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16. Redon C, Pilch DR, Rogakou EP, Orr AH, Lowndes NF, Bonner WM. Yeast histone 2A serine 129 is essential for the efficient repair of checkpoint-blind DNA damage. EMBO Rep 2003; 4:678-84. 17. Melo JA, Cohen J, Toczyski DP. Two checkpoint complexes are independently recruited to sites of DNA damage in vivo. Genes Dev 2001; 15:2809-21. 18. van den Bosch M, Bree RT, Lowndes NF. The MRN complex: coordinating and mediating the response to broken chromosomes. EMBO Rep 2003; 4:844-9. 19. Lukas C, Falck J, Bartkova J, Bartek J, Lukas J. Distinct spatiotemporal dynamics of mammalian checkpoint regulators induced by DNA damage. Nat Cell Biol 2003; 5:255-60. 20. Schwartz MF, Lee SJ, Duong JK, Eminaga S, Stern DF. FHA domain-mediated DNA checkpoint regulation of Rad53. Cell Cycle 2003; 2:384-96 21. Schwartz MF, Duong JK, Sun Z, Morrow JS, Pradhan D, Stern DF. Rad9 phosphorylation sites couple Rad53 to the Saccharomyces cerevisiae DNA damage checkpoint. Mol Cell 2002; 9:1055-65. 22. Fung KL, Hilgenberg L, Wang NM, Chirico WJ. Conformations of the nucleotide and polypeptide binding domains of a cytosolic Hsp70 molecular chaperone are coupled. J Biol Chem 1996; 271:21559-65. 23. Toh GW, Lowndes NF. Role of the Saccharomyces cerevisiae Rad9 protein in sensing and responding to DNA damage. Biochem Soc Trans 2003; 31:242-6. 24. de la Torre-Ruiz MA, Green CM, Lowndes NF. RAD9 and RAD24 define two additive, interacting branches of the DNA damage checkpoint pathway in budding yeast normally required for Rad53 modification and activation. EMBO J 1998; 17:2687-98.

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