p53 Transcriptional Activation Domain

[Cell Cycle 5:5, 489-494, 1 March 2006]; ©2006 Landes Bioscience

p53 Transcriptional Activation Domain Perspective

A Molecular Chameleon?

ABSTRACT The recent structure of human replication protein A (RPA) bound to residues 38–58 of tumor suppressor p53 exemplifies several important features of protein-protein interactions involved in transcription and DNA repair. First, the N-terminal transcriptional activation domain (TAD) of p53 is multifunctional and dynamic, showing multiple interactions with partner proteins some of which are modulated by phosphorylation. Second, the binding of partner proteins is coupled with a disorder-to-order transition common to many other transcriptional activation domains. Third, the molecular features of p53 residues 47-58 imitate those of single stranded DNA in their interaction with the oligonucleotide oliogsaccharide-binding (OB) fold of the N-terminal domain of RPA70. This regulated association is implicated in transmitting the DNA damage signal to the p53 pathway of stress response. Here we review the recently reported crystal structure of the p53/ RPA70N complex and the mechanism by which ssDNA can provide positive feedback to dissociate p53/RPA complexes. The binding mode and regulatory mechanisms of the p53/RPA70N interaction may represent a general paradigm for regulation of the OB folds involved in DNA repair and metabolism.

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Wild-type p53 is a nuclear tumor suppressor phosphoprotein. Inactivation of p53 is a key step in well over half of human cancers.1,2, The major role of p53 in normal cells is to induce cell cycle arrest or apoptosis in response cellular stress, particularly DNA damage.3 The primary mechanism by which p53 triggers these cellular outcomes is through its role as a sequence-specific DNA-binding transcription factor of genes involved in the regulation of the cell cycle and/or apoptosis which prevent damaged DNA from being replicated, emphasizing a role for p53 in maintaining the integrity of the genome.4 This DNA-damage checkpoint activity is a central to the role of p53 as a tumor suppressor; a detailed understanding of the p53 pathway crucial not only to understanding of cancer in general, but also has direct relevance to therapeutic strategies.5 Under normal conditions p53 is maintained at low levels by the cellular protein mdm2, which binds to p53,6 shuttles it out of the nucleus7 and acts as a ubiquitin E3 ligase8 to target p53 degradation. After DNA damage or stress, a series of post-translational modifications prevent the interaction of mdm2 with p53 and enhances the ability of p53 to bind sequence-specifically to p53-responsive promoters to activate transcription. Phosphorylation appears to play a central role in modulating p53 activity. All of the post-translational modifications of p53 occur in unstructured or natively disordered regions of the protein. Namely, the first 90 residues including the transactivation domain (TAD), the linker between DNA-binding domain and TET domain, and the C-terminal 30 residues. These regions also mediate p53’s interactions with many other proteins. Therefore, it is important to elucidate the dynamic structural details of these regions and their interactions to fully understand p53 regulation. Recently significant progress has been made in understanding the multiple ways in which the TAD can interact with protein partners. P53 has been reported to bind a number of proteins involved in DNA-repair or DNA-damage checkpoints. Among these is the interaction with replication protein A (RPA), the major single-stranded (ss)DNA binding protein of the eukaryotic nucleus.9-11 The interaction of p53 with RPA mediates suppression of homologous recombination12 and has been suggested to participate in the coordination of DNA repair with the p53dependent checkpoint control by sensing UV damage.13 Complex formation between p53

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Previously published as a Cell Cycle E-publication: http://www.landesbioscience.com/journals/cc/abstract.php?id=2489

KEY WORDS

INTRODUCTION

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Original manuscript submitted: 11/30/05 Manuscript accepted: 01/10/06

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*Correspondence to: Cheryl H. Arrowsmith; Ontario Cancer Institute and Department of Medical Biophysics; University of Toronto; 610 University Avenue; Toronto, Ontario M5G 2M9 Canada; Tel.: 416.946.0881; Fax: 416.946.0880; Email: [email protected]/ Alexey Bochkarev; Banting and Best Department of Medical Research & Department of Medical Genetics and Microbiology; University of Toronto; 112 College Street; Toronto, Ontario M5G 1L6 Canada; Tel.: 416.946.0805; Fax: 416.946.0588; Email: [email protected]

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1Ontario Cancer Institute and Department of Medical Biophysics; 2Banting and Best Department of Medical Research & Department of Medical Genetics and Microbiology; University of Toronto; Toronto, Ontario Canada

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Lilia Kaustov1 Gwan-Su Yi1 Ayeda Ayed1 Elena Bochkareva2 Alexey Bochkarev2,* Cheryl H. Arrowsmith1,2,*

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p53, transactivation domain, RPA, protein-protein interaction, X-ray, NMR

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ACKNOWLEDGEMENTS

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This work was supported by research grants from the National Institute of General Medical Science (to A.B.), the National Cancer Institute of Canada (NCIC) with Funds from the Canadian Cancer Society (to C.H.A.). A.A. was the recipient of a fellowship from the NCIC and L.K. is the recipient of a fellowship from the Leukemia and Lymphoma Society of Canada.

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p53 Transcriptional Activation Domain

and RPA can interfere with p53’s ability to bind A sequence specifically to DNA and is regulated by at 14 least two mechanisms. Firstly, it is regulated by the presence of various lengths of ssDNAs, as RPA, when bound to these DNAs, is unable to interact with p53. Secondly, UV radiation of cells greatly reduces the ability of RPA to bind to p53. B DNA-damage-induced hyperphosphorylated RPA does not associate with p53.13 RPA is a hetero-trimeric protein involved in both DNA replication and DNA repair.15,16 It binds to ssDNA and serves as a scaffold for the assembly of the DNA repair machinery. The largest subunit, RPA70, is a tandem repeat of four OB Figure 1. Residues 38-58 of p53 bind to RPA70N and form two helices. 15N-labeled and [oligonucleotide/oligosaccharide-binding]-folds;17 15N/13C labeled p53(1-72) were titrated with unlabelled RPA70(1-168) and RPA70(1-120). RPA70N, which is important for protein-protein Backbone resonances were assigned for most of the residues in free and bound p53(1-72). The interactions, and three ssDNA binding domains, resonance frequencies of the bound forms of p53(1-72) were identical for both RPA70(1-168) contribute RPA70A, RPA70B and RPA70C. RPA A and B or RPA(1-120) indicating that residues 121 through 168 of RPA70 do not significantly to the interaction. (A) Changes in chemical shift (as a weighted average of 15N and 1H values) 18 bind tightly to ssDNA and RPA70N has been for p53(1-72) upon binding to RPA70(1-168). (B) ∆(Cα-Cβ) shows the deviation from shown to interact weakly with DNA and p53.19 In residue-dependant random coil values of Cα and Cβ of p53(38-58) when bound to RPA70 response to DNA damage, at least three PI3 related (1-120). Consecutive positive or negative values identify α-helical or β-sheet regions respectively. kinases, ATM, ATR and DNA-PK hyper-phospho- The changes in resonance frequency for p53 residues 38-58 indicate the formation of two rylate the N-terminal domain of RPA32 α-helixes (positive values) upon binding to RPA(1-120). (RPA32N).20 Hyperphosphorylated, but not of RPA.25 By comparing the NMR spectra of a 15N-labeled p53 unphosphorylated RPA32N, is capable of binding RPA70N.21 Here we review the recently reported molecular interface between peptide (residues 1–70) before and after binding to RPA70N we part of the p53 TAD and RPA70N and discuss its implications for localized the RPA-binding region to residues 38–57 (Fig. 1). For the understanding the dynamic nature of the p53 TAD, the p53 first time we were able to directly observe changes in resonance DNA-damage response, and the broader mechanism of the OB fold frequency for p53 upon binding to RPA70N. The changes for p53 interactions with nucleic acids and proteins. p53 appears to directly residues 38–57 in complex with RPA70 (1–120) indicate the forinteract with RPA with the N terminal 70 residues of p53 (TAD) mation of two α-helices. This result agrees well with the region binding the N-terminal third of RPA70N, with some contribution previously mapped by scanning mutagenesis (residues 20–60),13 and from multiple sub-domains at the C-terminus.10,11,22 In RPA, the shows that other regions of the p53 TAD are not involved.26 binding requires some contribution from the C-terminal third as well.23 Mutational analysis indicated that aromatic amino acids STRUCTURE OF THE RPA70N/p53N COMPLEX Trp53 and Phe54 of p53 flanked by negatively charged residues are The X-ray crystallographic structure of a chimera comprising important for binding.1,22 However, relative contribution of these determinants was not clearly characterized. The crucial determinants residues 1–120 of RPA70 followed by residues 33–60 of p53 has 26 of the regulation of this protein-protein interaction are the OB-fold been recently solved at 1.6 Å resolution. The p53 peptide binds in the basic cleft of the RPA70N OB fold and binding induces significant domain of RPA70N and residues 37–57 of p53, a region associated conformational changes in the RPA70N. Both, electrostatic and with induction of apoptosis in response to UV damage. This study role in this interaction. interactions play an important hydrophobic suggests model in which ssDNA, a primary product of DNA Consistent with our NMR results p53 is folded into two helices, damage, can provide positive feedback to dissociate p53/RPA complexes, thus releasing each protein to function independently. H1 (residues 41–44) and H2 (residues 47–55) with a linker The OB-fold of RPA70N thus represents a novel structural entity (residues 45–46) that makes a sharp turn such that the p53 helices endowing dual affinity (1) for acidic, amphipathic peptide helices appear to wrap around RPA. Helix H2 has the most extensive buried surface area and, therefore, appears to be the major determinant of and (2) for ssDNA. the interaction. It binds in the deep basic cleft corresponding to the nucleic acid-binding pocket of the OB fold. In solution, the most RPA70N INDUCES HELICAL STRUCTURE IN A SUBREGION likely mode of interaction is that observed for the H2. Helix 1 has a OF THE p53 TRANSACTIVATION DOMAIN smaller interaction surface and smaller changes in NMR resonance The N-terminal domain of RPA70 (residues 1–169) is a putative frequencies upon binding to RPA70N, suggesting that H1 plays a protein-protein interaction domain.24 Its NMR structure24 contains secondary role in the interaction (Fig. 2). a large basic cleft with an extensive positively charged surface. The RPA/p53 interface has similarities with those of the MDM2 Previous reports demonstrated that the amino terminal acidic region oncoprotein bound to the p53 transactivation domain (residues of p53, particularly amino acids 20 to 73, is the major determinant 17–29).27 In both structures, the interface is mediated by two aromatic of RPA70N binding with additional affinity provided by C-terminal and one hydrophobic residues of p53 (Phe-19, Trp-23 and Leu-26 residues.9,13,22 in MDM2/p53). For both interactions, p53 undergoes a Recently, Vise et al. using NMR spectroscopy further narrowed down coil-to-helix transition upon binding. However, despite these gross the region of interaction to residues 39–59 of p53 and residues 1–168 similarities, RPA does not bind the primary MDM2 binding site of 490

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groups of DNA exposed to the solvent and the aromatic bases are involved in stacking or hydrophobic interactions with the base of the binding cleft (Fig. 3A). The same interaction mode is observed in RPA70N-p53 complex (Fig. 3B), where five negatively charged side chains are exposed to solvent and two aromatic residues, Trp-53 and Phe-54 are homologous of bases C1 and C2 in the RPA70A/ssDNA complex. Characteristically, flexibility of the RPA70N loops mediates ssDNA binding in the case of the RPA and BRCA2 OB folds.16,29

ssDNA AND PHOSPHOMIMETIC PEPTIDE OF RPA32 COMPETE WITH p53N FOR BINDING TO RPA70N

Daughdrill et al.19 has shown that the RPA70N can interact weakly with ssDNA. The RPA70N residues with the largest chemical shift changes after addition of ssDNA cluster near the basic cleft that is also important for RPA70N-protein interactions. NMR chemical shift mapping suggested that ssDNA interacts with a region that largely overlaps with the p53 binding site of RPA70N. Furthermore, Figure 2. Structure of RPA70N/p53N complex. RPA70N molecules interacts it is known that ssDNA disrupts the RPA70-p53 interaction.14 with two separate p53 peptides. Two separate H1 helices, one from the Based on these two observations it was hypothesized that p53 and internal fusion partner of RPA70N (blue) and other (gold) from a neighboring ssDNA are in direct competition for the binding site in RPA70N. molecule, interact with two separate sites of RPA. The RPA70N is in green. Bochkareva et al.26 used 15N-HSQC NMR titration experiments to Helix H2 is a major determinant of the interaction. H1 and H2 are helices demonstrate competition between ssDNA and p53 thereby conwithin p53. firming the hypothesis that ssDNA and p53 are in direct competition for a binding site on the RPA70N. A direct interaction between the basic cleft B A of RPA70N and the peptide corresponding to the negatively charged (RPA32-Asp) phosphorylation domain has been reported, whereas a peptide corresponding to the native, unphosphorylated sequence did not, suggesting a possibility for yet another competition.30 NMR titration experiments were again used to test whether such a peptide can compete with p53 for binding to RPA70N. A hyperphosphorylation mimetic peptide (RPA32NASP) in which Ser/Thr residues that have been reported to be phosphorylated in vivo (Ser8, Ser13, Thr21, Ser23, Ser29 and Ser33)31 were replaced with Asp residues that mimic phosphoserine. In addition to nine serines/threonines, at least six of which are phosphorylated in vivo, this peptide also contains six aromatic residues and could potentially form one or more amphipathic helices. The binding affinities of the three species Figure 3. Structural mimicry in the RPA70N/p53 and RPA70N/ssDNA complexes.(A) Structure of for RPA70N were estimated to be in the range RPA70A (ribbon diagram) bound to ssDNA. Bases C1 and C2 mediate interaction in the binding cleft of 10–150 µM, with ssDNA having ~10-fold and acidic phosphates exposed to solvent. (B) Aromatic side chains of p53 helix H2 mediate the lower affinity than p53N and RPA32Asp interaction with the binding cleft of RPA70N and acidic side chains are exposed to solvent. having an intermediate affinity. The OB-fold of RPA70N, although structurally similar to p53 (residues 17–29).25,27 Interestingly, Chi et al.28 have recently the DNA-binding domains of RPA, lacks two conserved aromatic shown that residues 40–45 and 49–54 of p53 are also capable of residues characteristic of the other ssDNA-binding domains.16 This binding to mdm2 indicating that mdm2 has dual specificity for may explain in part, why RPA70N has a much lower affinity for N-terminal regions of p53. However, this does not appear to be the ssDNA, and is also capable of binding negatively charged but case for RPA as shown in Figure 1. amphipathic peptides such as p53, and hyperphosphorylated The mode of p53 binding to RPA is similar to that of ssDNA RPA32N. binding to the DNA-binding domains of RPA.18 In the RPA70AThis data indicates that a negatively charged RPA32N-Asp peptide ssDNA complex, DNA binding is mediated by negative phosphate (mimicking that after DNA damage) can effectively compete with www.landesbioscience.com

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p53 for binding to RPA70N suggesting that RPA phosphorylation modulates RPA mediated interaction with DNA and proteins thereby causing modulation of RPA activity.

p53 TAD: DYNAMIC MULTIFUNCTIONAL DOMAIN

Our NMR studies on p53 TAD (1–70) showed that it is largely disordered under physiological conditions. Three small regions within the TAD have been shown to have minimally-structured motifs, a helix and two nascent turns although these are not static globular structures.32 A helix formed by residues 18–26 can be most clearly identified and preexists even in the absence of any target protein. This helix coincides with the amphipathic helix that was reported to be induced upon mdm2 binding.27,33 Recently, two additional mini-motifs (turn I, residues 40–45 and turn II, 49–54) were shown also to bind to mdm2.28 In particular, the turn II motif has a higher mdm2-binding affinity than the turn I and targets the same site in mdm2 as the helix. These motifs correspond directly with the RPA-binding helices H1 and H2 of p53 and suggest that this region of p53 may be multifunctional in its ability to interact with multiple protein partners. The ability of intrinsically unstructured proteins (IUP) to participate in the complex regulatory processes is becoming a new paradigm in molecular biology. It is hypothesized that IUP’s are able to remodel their structures to interact with multiple protein partners. Our data strongly support a mechanism where the folding of p53TAD is coupled to binding RPA70N.26 In the case of p53mdm2 complex, Chi et al.28 proposed that the presence of the turns in p53 TAD could increase the initial mdm2-bound fraction of p53 compared to the case where only the helix motif exists in p53 TAD, due to a small fraction of a turn-bound mdm2 which may eventually convert to a more stable helix -bound complex. The well-known fact that several other activation domains also contain similar multiple hydrophobic mini-domains capable of forming local minimallystructured motifs suggests that what has been found in p53 TAD corresponds to a general structural and functional feature common to other transcriptional activation domains or factors.34-38 It should be mentioned that such minimally structured functional motifs in p53 are all present within the TAD where the majority of phosphorylation sites are also frequently clustered (Fig. 4). p53 phosphorylation on several serine residues mostly within the N-terminal domain, which includes the mdm2 binding site, has been considered critical not only for inhibition of its interaction with mdm2 but also for its activation as a transcription factor.39-41 Recent studies point out that the sites of structural disorder and phosphorylation are highly correlated.42 Conformational changes upon phosphorylation often affect protein function. For example, serine phosphorylation of the peptide corresponding to the calmodulin binding domain of human protein p4.1 influences the ability of the peptide to adopt an α-helical conformation and thereby impairs the calmodulin-peptide interaction.43 Another example is the v-cyclin-CDK6-mediated phosphorylation of two serines in the unstructured loop of Bcl-2, which abolishes its antiapoptotic potential.44 To gain further insight regarding the role of disorder in the phosphorylation process, Iacoucheva et al.42 have investigated more than 1500 experimentally determined phosphorylation sites in eukaryotic proteins and compared them with ordered and disordered protein regions. They observed that the similarity in sequence complexity, amino acid composition, flexibility parameters, and other properties between phosphorylation sites and disordered protein regions suggests that intrinsic disorder 492

Figure 4. Structure and posttranslational modifications of the p53 transactivation domain, TAD, and its complexes with mdm2 and RPA. Boxed numbers represent the Ser/Thr phosphorylation sites. 17 serine and threonine sites have been reported to be phosphorylated on p53. Most of the sites are located in the amino-terminal region.

in and around the potential phosphorylation target site is an essential common feature for eukaryotic serine, threonine and tyrosine phosphorylation sites. The tumor suppressor p53 plays a central role in preserving genomic integrity by arresting cell cycle progression or activating apoptosis after genotoxic stress.2,45 Numerous studies have demonstrated that the activity of p53 protein is modulated by post-translational modifications including the phosphorylation of specific residues in the N- and C-terminal domains.39 The RPA-binding region of p53 contains two functionally important phosphorylation sites, Ser 37 and Ser 46. Phosphorylation of p53 at serine 37 is involved in the response to DNA damage.46,47 The structure of the R PA70N-p53 complex suggests that phosphorylation of Ser 37 would disrupt its hydrogen bonding interaction with Asp 89 of RPA70.26 Thus, phosphorylation of p53 at this site could be an additional mechanism to regulate the p53-RPA interaction. Ser 46 is phosphorylated in response to UV damage and is associated with p53-mediated apoptosis.46,48 Ser 46 is in the linker area that connects helices H1 and H2 and makes a turn such that p53 helices appear to wrap around RPA. Phosphorylation of these residues could potentially modulate the p53-RPA interaction. Alternatively, because of the specific conformation of the p53 peptide when bound to RPA, the ability of Ser-37 and/or Ser-46 to interact productively with a kinase active site might be affected. It should be mentioned that Ser 46 is exposed to solvent, suggesting that phosphorylation of this residue would not necessarily disrupt the p53/RPA interaction. Indeed NMR and flourescence-based binding assays with a synthetic Ser 46 phosphopeptide showed no significant affect on p53 binding to RPA70N (data not shown). This suggests that the p53 RPA70N interaction is not regulated by phosphorylation at Ser46. Considering the importance of Ser46 phosphorylation in apoptosis, along with the involvement of Ser46 phosphorylation in disrupting of mdm2 interaction, phosphorylation of this site may play a critical role for separating p53 from mdm2.49

MOLECULAR MIMICRY: TRICKING THE OB FOLD

Crucial to the regulation of the p53-RPA interaction is the OB-fold domain of RPA70N. The structural and functional data also indicate a mechanism by which ssDNA, a primary product of

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DNA damage, can provide positive feedback to dissociate p53/RPA complexes, thus releasing each protein to function independently. Bochkareva et al50 proposed that p53 (and many other proteins) interact with RPA via a “two-point interaction” in which two distinct areas of the p53 (p53N and p53C) contact two distinct areas of RPA (RPA70N and RPA70C). After DNA binding, the N- and C-termini of RPA70 move apart, allosterically switching the two-point interaction. Vise et al proposed a more complex25 model in which ssDNA binding by RPA70A and RPA70B positions RPA70N into an orientation favorable for binding ssDNA. The linkage effect increases the affinity of RPA70N to ssDNA by increasing the local concentration of ssDNA thereby effectively competing with p53.25 Our new data is in agreement with this model and provides it with a structural basis.26 The details of the interaction between RPA70C and the C-terminal domain of p53 remain to be elucidated. It is generally believed that the overall cellular response to DNA damage is in part determined by the degree of damage sustained. DNA damage induces hyperphosphorylation of RPA32.20,51,52,53 Our model suggest a possible role for RPA32N in the regulation of RPA70N binding to either p53 or ssDNA. We show that a peptide mimetic of phosphorylated RPA32N is able to dissociate a p53 peptide from its OB binding site on RPA70N. Thus, it is possible that ssDNA and/or phosphorylated RPA32, both consequences of DNA damage, can structurally compete with p53 (and one another) for binding to the OB fold of RPA70N. The OB fold of RPA70N thus represents a structural entity endowing dual affinity (1) for acidic, amphipathic peptide helices and (2) for ssDNA. Binding is mutually exclusive and may be hierarchical. The binding mode and regulatory mechanisms of the RPA-p53 interaction may represent a general paradigm for regulation of the OB folds involved in DNA repair and metabolism. For example, the N-terminal acidic peptide of Rad51 and ssDNA compete for binding to the ssDNA-binding domain, RPA70A, which is a canonical OB fold.54 The interaction between RPA70B and hyperphosphorylated RPA32N has been proposed to play a role in modulating the cellular pathways by altering the RPA70B-mediated RPA-DNA and RPAprotein interactions.55 The acidic transactivation domain of BRCA2 binds RPA. Although the precise domain of RPA has not been mapped, the cancer predisposing mutation of Tyr-42 to Cys (aromatic residue) significantly compromises this interaction.56 Many other acidic transactivators may share the same regulatory mechanisms, and many other OB folds and those of RPA may also be subject to similar competition between proteins and ssDNA. The purpose of RPA phosphorylation has remained poorly understood since it does not detectably affect ssDNA binding—the major functional role of RPA.57 Bochkareva et al.26 have shown that RPA phosphorylation can serve as a mediator of RPA-protein interactions, especially with p53. The pattern of RPA phosphorylation differs depending on the cell cycle and DNA damage. The effect of different phosphorylation signals on RPA-protein interaction remains to be understood. The combined effect of the ssDNA and phosphorylated RPA32N displaces the p53 peptide out of the binding site and, may thereby expose p53 for phosphorylation. Thus, a consequence of the interaction could be the masking of phosphorylation sites at Ser37 and Ser46 of p53 and delayed phosphorylation by kinases that act upon Ser37 and Ser46 until after release by RPA, with RPA32N phosphorylation serving as a “safe-proof ” mechanism that ensures p53 would be activated only at sites of DNA damage, and not as a result of an accidental signal. Clearly other factors may further embellish www.landesbioscience.com

this crucial network of protein-protein and protein-ssDNA interactions. Any such changes carry the potential for contributing to initiation of “sensor networking” regulated by p53 and RPA following DNA damage. References 1. Ko LJ, Prives C. p53: Puzzle and paradigm. Genes Dev 1996; 10:1054-72. 2. Levine AJ. P53, the cellular gatekeeper for growth and division. Cell 1997; 88:323-31. 3. Kastan MB, Zhan Q, El-Deiry WS, Carrier F, Jacks T, Walsh WV, Plunkett BS, Vogelstein B, Fornace AJ. A mammalian cell cycle checkpoint pathway utilizing p53 and GADD45 is defective in ataxia-telangiectasia. Cell 1992; 71:587-97. 4. Lane DP. P53, guardian of the genome. Nature 1992; 358:15-6. 5. Vassilev LT. P53 activation by small molecules: Application in oncology. J Med Chem 2005; 48:4491-9. 6. Midgley CA, Lane DP. P53 protein stability in tumor cells is not determined by mutation but is dependent on mdm2 binding. Oncogene 1997; 15:1179-89. 7. Roth J, Dobblestein M, Freedman DA, Shenk T, Levine AJ. Nucleo-cytoplasmic shuttling of the mdm2 oncoprotein regulates the levels of the p53 protein via a pathway used by the human immunodeficiency virus rev protein. EMBO J 1998; 17:554-64. 8. Honda R, Tanaka H, Yasuda H. Oncoprotein mdm2 is a ubiquitin ligase for tumor suppressor p53. Science 1998; 281:1677-9. 9. Dutta A, Ruppert JM, Aster JC, Winchester E. Inhibition of DNA replication factor RPA by p53. Nature 1993; 365:79-82. 10. He Z, Brinton BT, Greenblatt J, Hassell JA, Ingles CJ. The transactivator proteins VP16 and GAL4 bind replication factor A. Cell 1993; 73:1223-32. 11. Li R, Botchan MR. The acidic transcriptional activation domains of VP16 and p53 bind the cellular replication protein A and stimulate in vitro BPV-1 DNA replication. Cell 1993; 73:1207-21. 12. Romanova LY, Willers H, Blagosklonny MV, Powell SN. The interaction of p53 with replication protein A mediates suppression of homologous recombination. Oncogene 2004; 23:9025-33. 13. Abramova NA, Russell J, Botchan M, Li R. Interaction between replication protein A and p53 is disrupted after UV damage in a DNA repair-dependent manner. Proc Natl Acad Sci USA 1997; 94:7186-91. 14. Miller SD, Moses K, Jayaraman L, Prives C. Complex formation between p53 and replication protein A inhibits the sequence-specific DNA binding of p53 and is regulated by single-stranded DNA. Mol Cell Biol 1997; 17:2194-2201. 15. Wold MS. Replication protein A: A heterotrimeric, single-stranded DNA-binding protein required for eukaryotic DNA metabolism. Annu Rev Biochem 1997; 66:61-92. 16. Bochkarev A, Bochkareva E. From RPA to BRCA2: Lessons from single-stranded DNA binding by the OB-fold. Curr Opin Struct Biol 2004; 14:36-42. 17. Murzin AG. OB(oligonucleotide/oligosaccharide binding)-fold: Common structural and functional solution for nonhomologous sequences. EMBO J 1993; 12:861-7. 18. Bochkarev A, Pfuetzner RA, Edwards AM, Frappier L. Structure of the single-strandedDNA-binding domain of replication protein A bound to DNA. Nature 1997; 385:176-81. 19. Daughdrill GW, Ackerman J, Isern NG, Botuyan MV, Arrowsmith C, Wold MS, Lowry DF. The weak interdomain coupling observed in the 70 kDa subunit of human replication protein A is unaffected by ssDNA binding. Nucleic Acids Res 2001; 29:3270-6. 20. Block WD, Yu Y, Lees-Miller SP. Phosphatidyl inositol 3-kinase-like serine/threonine protein kinases (PIKKs) are required for DNA damage-induced phosphorylation of the 32 kDa subunit of replication protein A at threonine 21. Nucleic Acids Res 2004; 32:997-1005. 21. Binz SK, Sheehan AM, Wold MS. Replication protein A phosphorylation and the cellular response to DNA damage. DNA Repair (Amst) 2004; 3:1015-24. 22. Leiter LM, Chen J, Marathe T, Tanaka M, Dutta A. Loss of transactivation and transrepression function, and not RPA binding, alters growth suppression by p53. Oncogene 1996; 12:2661-68. 23. Lin YL, Chen C, Keshav KF, Winchester E, Dutta A. Dissection of functional domains of the human DNA replication protein complex replication protein A. J Biol Chem 1996; 271:17190-8. 24. Jacobs DM, Lipton AS, Isern NG, Daughdrill GW, Lowry DF, Gomes X, Wold MS. Human replication protein A: Global fold of the N-terminal RPA-70 domain reveals a basic cleft and flexible C-terminal linker. J Biomol NMR 1999; 14:321-31. 25. Vise PD, Baral B, Latos AJ, Daughrill GW. NMR chemical shift and relxation measurements provide evidence for the coupled folding and binding of the p53 transactivation domain. Nuc Acid Res 2005; 33:2061-77. 26. Bochkareva E, Kaustov L, Ayed A, Yi GW, Lu Y, Pineda-Lucena A, Liao JCC, Okorokov AL, Milner J, Arrowsmith CH, Bochkarev A. Single-stranded DNA mimicry in the p53 transactivation domain interaction with replication protein A. Proc Natl Acad Sci USA 2005; 102:15412-7. 27. Kussie PH, Gorina S, Marechal V, Elenbaas B, Moreau J, Levine AJ, Pavletich NP. Structure of the MDM2 oncoprotein bound to the p53 tumor suppressor transactivation domain. Science 1996; 274:948-53. 28. Chi SW, Lee SH, Kim DH, Ahn MJ, Kim JS, Woo JY, Torizawa T, Kainosho M, Han KH. Structural details on mdm2-p53 interaction. J Biol Chem 2005; 280:38795-802. 29. Yang H, Jeffrey PD, Miller J, Kinnucan E, Sun Y, Thoma NH, Zheng N, Chen PL, Lee WH, Pavletich NP. BRCA2 function in DNA binding and recombination from a BRCA2-DSS1-ssDNA structure. Science 2002; 297:1837-48.

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493


30. Binz SK, Lao Y, Lowry DF, Wold MS. The phosphorylation domain of the 32-kDa subunit of replication protein A (RPA) modulates RPA-DNA interactions. Evidence for an intersubunit interaction. J Biol Chem 2003; 278:35584-91. 31. Zernik-Kobak M, Vasunia K, Connelly M, Anderson CW, Dixon K. Sites of UV-induced phosphorylation of the p34 subunit of replication protein A from HeLa cells. J Biol Chem 1997; 272:23896-904. 32. Lee H, Mok KH, Muhandiram R, Park KH, Suk JE, Kim DH, Chang J, Sung YC, Choi KY, Han KH. Local structural elements in the mostly unstructured transcriptional activation domain of human p53. J Biol Chem 2000; 275:29426-32. 33. Botuyan MV, Momand J, Chen Y. Solution conformation of an essential region of the p53 transactivation domain. Fold Des 1997; 2:331-42. 34. Dyson HJ, Wright PE. Coupling of folding and binding for unstructured proteins. Curr Opin Struct Biol 2002; 12:54-60. 35. Wright PE, Dyson HJ. Intrinsically unstructured proteins: Reassessing the protein structurefunction paradigm. J Mol Biol 1999; 293:321-31. 36. Regier JL, Shen F, Triezenberg SJ. Pattern of aromatic and hydrophobic amino acids critical for one of two subdomains of the VP16 transcriptional activator. Proc Natl Acad Sci USA 1993; 90:883-7. 37. Metivier R, Penot G, Flouriot G, Pakdel F. Synergism between ERalpha transactivation function 1 (AF-1) and AF-2 mediated by steroid receptor coactivator protein-1: Requirement for the AF-1 alpha-helical core and for a direct interaction between the N- and C-terminal domains. Mol Endocrinol 2001; 15:1953-70. 38. Semenza GL. HIF-1 and mechanisms of hypoxia sensing Current Opin Cell Biol 2001; 13:167-71. 39. Bode AM, Dong Z. Post-translational modification of p53 in tumorigenesis. Nat Rev Cancer 2004; 4:793-805. 40. Pise-Masison CA, Radonovich M, Sakaguchi K, Appella E, Brady JN. Phosphorylation of p53: A novel pathway for p53 inactivation in human T-cell lymphotropic virus type 1-transformed cells. J Virol 1998;72:6348-55. 41. Brooks CL, Gu W. Ubiquitinqtion, phosphorylation and acetylation: The molecular basis for p53 regulation. Curr Opin Cell Biol 2003; 15:164-71. 42. Iakoucheva LM, Radivojac P, Brown CJ, O’Connor TR, Sikes JG, Obradovic Z, Dunker AK. The importance of intrinsic disorder for protein phosphorylation. Nucleic Acid Res 2004; 32:1037-49. 43. Vetter SW, Leclerc E. Phosphorylation of serine residues affects the conformation of the calmodulin binding domain of human protein 4.1. Eur J Biochem 2001; 268:4292-99. 44. Ojala PM, Yamamoto K, Castanos-Velez E, Biberfeld P, Korsmeyer SJ, Makela TP. The apoptotic v-cyclin-CDK6 complex phosphorylates and inactivates Bcl-2. Nature Cell Biol 2000; 2:819-25. 45. Prives C, Hall PA. The p53 pathway. J Pathol 1999; 187:112-26. 46. Bulavin DV, Saito S, Hollander MC, Sakaguchi K, Anderson CW, Appella E, Fornace AJ. Phosphorylation of human p53 by p38 kinase coordinates N-terminal phosphorylation and apoptosis in response to UV radiation. EMBO J 1999; 18:6845-54. 47. Dohoney KM, Guillerm C, Whiteford C, Elbi C, Lambert PF, Hager GL, Brady JN. Phosphorylation of p53 at serine 37 is important for transcriptional activity and regulation in response to DNA damage. Oncogene 2004; 23:49-57. 48. Oda K, Arakawa H, Tanaka T, Matsuda K, Tanikawa C, Mori T, Nishimori H, Tamai K, Tokino T, Nakamura Y, Taya Y. p53AIP1, a potential mediator of p53-dependent apoptosis, and its regulation by Ser-46-phosphorylated p53. Cell 2000; 102:849-62. 49. Di Stefano V, Blandino G, Sacchi A, Soddu S, D’Orazi G. HIPK2 neutralizes MDM2 inhibition rescuing p53 transcriptional activity and apoptotic function. Oncogene 2004; 23:5185-92. 50. Bochkareva E, Belegu V, Korolev S, Bochkarev A. Structure of the major single-stranded DNA-binding domain of replication protein A suggests a dynamic mechanism for DNA binding. EMBO J 2001; 20:612-8. 51. Liu VF, Weaver DT. The ionizing radiation-induced replication protein A phosphorylation response differs between ataxia telangiectasia and normal human cells. Mol Cell Biol 1993; 13:7222-31. 52. Brush GS, Anderson CW, Kelly TJ. The DNA-activated protein kinase is required for the phosphorylation of replication protein A during simian virus 40 DNA replication. Proc Natl Acad Sci USA 1994; 91:12520-4. 53. Brush GS, Morrow DM, Hieter P, Kelly TJ. The ATM homologue MEC1 is required for phosphorylation of replication protein A in yeast. Proc Natl Acad Sci USA 1996; 93:15075-80. 54. Stauffer ME, Chazin WJ. Physical interaction between replication protein A and Rad51 promotes exchange on single-stranded DNA. J Biol Chem 2004; 279:25638-45. 55. Liu Y, Kvaratskhelia M, Hess S, Qu Y, Zou Y. Modulation of replication protein A function by its hyperphosphorylation-induced conformational change involving DNA binding domain B. J Biol Chem 2005; 280:32775-83. 56. Wong JM, Ionescu D, Ingles CJ. Interaction between BRCA2 and replication protein A is compromised by a cancer-predisposing mutation in BRCA2. Oncogene 2003; 22:28-33. 57. Henricksen LA, Carter T, Dutta A, Wold MS. Phosphorylation of human replication protein A by the DNA-dependent protein kinase is involved in the modulation of DNA replication. Nucleic Acids Res 1996; 24:3107-12.

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