Recent Replication Papers

Origins of DNA replication in the three domains of life.

Robinson NP, Bell SD

FEBS J 272:3757-66
Aug , 2005

Replication of DNA is essential for the propagation of life. It is somewhat surprising then that, despite the vital nature of this process, cellular organisms show a great deal of variety in the mechanisms that they employ to ensure appropriate genome duplication. This diversity is manifested along classical evolutionary lines, with distinct combinations of replicon architecture and replication proteins being found in the three domains of life: the Bacteria, the Eukarya and the Archaea. Furthermore, although there are mechanistic parallels, even within a given domain of life, the way origins of replication are defined shows remarkable variation.

p53-Dependent Regulation of Cdc6 Protein Stability Controls Cellular Proliferation.

Duursma A, Agami R

Mol Cell Biol 25:6937-47
Aug , 2005

Activation of tumor suppressor p53 in response to genotoxic stress imposes cellular growth arrest or apoptosis. We identified Cdc6, a licensing factor of the prereplication complex, as a novel target of the p53 pathway. We show that activation of p53 by DNA damage results in enhanced Cdc6 destruction by the anaphase-promoting complex. This destruction is triggered by inhibition of CDK2-mediated CDC6 phosphorylation at serine 54. Conversely, suppression of p53 expression results in stabilization of Cdc6. We demonstrate that loss of p53 results in more replicating cells, an effect that can be reversed by reducing Cdc6 protein levels. Collectively, our data suggest that initiation of DNA replication is regulated by p53 through Cdc6 protein stability.

Apoptosis in budding yeast caused by defects in initiation of DNA replication.

Weinberger M, Ramachandran L, Feng L, Sharma K, Sun X, Marchetti M, Huberman JA, Burhans WC

J Cell Sci 118:3543-53
Aug 1, 2005

Apoptosis in metazoans is often accompanied by the destruction of DNA replication initiation proteins, inactivation of checkpoints and activation of cyclin-dependent kinases, which are inhibited by checkpoints that directly or indirectly require initiation proteins. Here we show that, in the budding yeast Saccharomyces cerevisiae, mutations in initiation proteins that attenuate both the initiation of DNA replication and checkpoints also induce features of apoptosis similar to those observed in metazoans. The apoptosis-like phenotype of initiation mutants includes the production of reactive oxygen species (ROS) and activation of the budding-yeast metacaspase Yca1p. In contrast to a recent report that activation of Yca1p only occurs in lysed cells and does not contribute to cell death, we found that, in at least one initiation mutant, Yca1p activation occurs at an early stage of cell death (before cell lysis) and contributes to the lethal effects of the mutation harbored by this strain. Apoptosis in initiation mutants is probably caused by DNA damage associated with the combined effects of insufficient DNA replication forks to completely replicate the genome and defective checkpoints that depend on initiation proteins and/or replication forks to restrain subsequent cell-cycle events until DNA replication is complete. A similar mechanism might underlie the proapoptotic effects associated with the destruction of initiation and checkpoint proteins during apoptosis in mammals, as well as genome instability in initiation mutants of budding yeast.

Regulation of mitosis in response to damaged or incompletely replicated DNA require different levels of Grapes (Drosophila Chk1).

Purdy A, Uyetake L, Cordeiro MG, Su TT

J Cell Sci 118:3305-15
Aug 1, 2005

Checkpoints monitor the state of DNA and can delay or arrest the cell cycle at multiple points including G(1)-S transition, progress through S phase and G(2)-M transition. Regulation of progress through mitosis, specifically at the metaphase-anaphase transition, occurs after exposure to ionizing radiation (IR) in Drosophila and budding yeast, but has not been conclusively demonstrated in mammals. Here we report that regulation of metaphase-anaphase transition in Drosophila depends on the magnitude of radiation dose and time in the cell cycle at which radiation is applied, which may explain the apparent differences among experimental systems and offer an explanation as to why this regulation has not been seen in mammalian cells. We further document that mutants in Drosophila Chk1 (Grapes) that are capable of delaying the progress through mitosis in response to IR are incapable of delaying progress through mitosis when DNA synthesis is blocked by mutations in an essential replication factor encoded by double park (Drosophila Cdt1). We conclude that DNA damage and replication checkpoints operating in the same cell cycle at the same developmental stage in Drosophila can exhibit differential requirements for the Chk1 homolog. The converse situation exists in fission yeast where loss of Chk1 is more detrimental to the DNA damage checkpoint than to the DNA replication checkpoint. It remains to be seen which of these two different uses of Chk1 homologs are conserved in mammals. Finally, our results demonstrate that Drosophila provides a unique opportunity to study the regulation of the entry into, and progress through, mitosis by DNA structure checkpoints in metazoa.

Control of replication initiation and heterochromatin formation in Saccharomyces cerevisiae by a regulator of meiotic gene expression.

Irlbacher H, Franke J, Manke T, Vingron M, Ehrenhofer-Murray AE

Genes Dev 19:1811-22
Aug 1, 2005

Heterochromatinization at the silent mating-type loci HMR and HML in Saccharomyces cerevisiae is achieved by targeting the Sir complex to these regions via a set of anchor proteins that bind to the silencers. Here, we have identified a novel heterochromatin-targeting factor for HML, the protein Sum1, a repressor of meiotic genes during vegetative growth. Sum1 bound both in vitro and in vivo to HML via a functional element within the HML-E silencer, and sum1Delta caused HML derepression. Significantly, Sum1 was also required for origin activity of HML-E, demonstrating a role of Sum1 in replication initiation. In a genome-wide search for Sum1-regulated origins, we identified a set of autonomous replicative sequences (ARS elements) that bound both the origin recognition complex and Sum1. Full initiation activity of these origins required Sum1, and their origin activity was decreased upon removal of the Sum1-binding site. Thus, Sum1 constitutes a novel global regulator of replication initiation in yeast.

Genomewide analysis of nucleosome density histone acetylation and HDAC function in fission yeast.

WirÃÂ©n M, Silverstein RA, Sinha I, Walfridsson J, Lee HM, Laurenson P, Pillus L, Robyr D, Grunstein M, Ekwall K

EMBO J :HASH(0x8bc130)
Aug 4, 2005

We have conducted a genomewide investigation into the enzymatic specificity, expression profiles, and binding locations of four histone deacetylases (HDACs), representing the three different phylogenetic classes in fission yeast (Schizosaccharomyces pombe). By directly comparing nucleosome density, histone acetylation patterns and HDAC binding in both intergenic and coding regions with gene expression profiles, we found that Sir2 (class III) and Hos2 (class I) have a role in preventing histone loss; Clr6 (class I) is the principal enzyme in promoter-localized repression. Hos2 has an unexpected role in promoting high expression of growth-related genes by deacetylating H4K16Ac in their open reading frames. Clr3 (class II) acts cooperatively with Sir2 throughout the genome, including the silent regions: rDNA, centromeres, mat2/3 and telomeres. The most significant acetylation sites are H3K14Ac for Clr3 and H3K9Ac for Sir2 at their genomic targets. Clr3 also affects subtelomeric regions which contain clustered stress- and meiosis-induced genes. Thus, this combined genomic approach has uncovered different roles for fission yeast HDACs at the silent regions in repression and activation of gene expression.

Mcm10 is Required for the Maintenance of Transcriptional Silencing in Saccharomyces cerevisiae.

Liachko I, Tye BK

Genetics :HASH(0x774860)
Aug 5, 2005

Mcm10 is an essential protein that participates in both the initiation and elongation of DNA replication. In this study we demonstrate a role for Mcm10 in the maintenance of heterochromatic silencing at telomeres and HM loci of budding yeast. Two mcm10 mutants drastically reduce silencing of both URA3 and ADE2 reporter genes integrated into these silent loci. When exposed to alpha-factor, mcm10 mutant cells display a "shmoo-cluster" phenotype associated with a defect in the maintenance of silencing. In addition, when combined with a defect in the establishment of silent chromatin, mcm10 mutants demonstrate a synergistic defect in HML silencing. Consistent with a direct silencing function, Mcm10p shows a two-hybrid interaction with Sir2p and Sir3p that is destroyed by the mcm10-1 mutation and dependent on the C-terminal 108 amino acids. Tethering GBD-MCM10 to a defective HMR-E silencer is not sufficient to restore silencing. Furthermore, mutations in MCM10 inhibit the ability of GBD-SIR3 to restore silencing when tethered to a defective HMR-E. Suppressor mutations in MCM2, which suppress the temperature sensitivity of mcm10-1, fail to overcome the mcm10-1 silencing defect suggesting that MCM10's role in transcriptional silencing may be separate from its essential functions in DNA replication.

DNA Replication Initiates at Different Sites in Early and Late S Phase within Human Ribosomal RNA Genes.

Coffman FD, He M, Diaz ML, Cohen S

Cell Cycle 4:HASH(0x8bdc78)
Sep 22, 2005

Metazoan replication origins often contain multiple potential initiation sites, and the selection of which of the potential sites are used appears to be dependent upon multiple factors, including the state of differentiation, cell metabolism, and local transcriptionaI activity. Numerous studies have shown that a replication origin exists within the non-transcribed spacer region of the human ribosomal RNA gene. We here analyze nascent leading strand DNA from S phase human lymphoid cells, and find that while the majority of rDNA replicates in mid- and late S phase and preferentially initiates replication 6 kbp from the transcription start site, in very early S phase the preferred initiation site is much closer to the transcription start site and may involve rDNA promoter sequences. This early site is coincident with a minimum GC skew value, diagnostic for replication origins in bacteria and yeast. These results suggest that replication timing can influence initiation site selection. The timing and nucleolar localization of rDNA further suggest that this site likely participates in the small number of perinucleolar initiation foci observed in very early S phase cells that represent the beginning of cellular DNA replication.

A Novel Cdk2 Interactor is Phosphorylated by Cdc7 and Associates with Components of the Replication Complexes.

Grishina I, Lattes B

Cell Cycle 4:HASH(0x8bd9b4)
Aug 10, 2005

Initiation of DNA replication in eukaryotic cells depends on the assembly of the prereplication complexes containing two hexamers, the Origin Recognition Complex (ORC) and the Minichromosome maintenance/DNA Replication Licensing complex (MCM); and on the subsequent conformational changes in the MCM complex leading to the formation of a competent DNA replication complex, firing of the DNA polymerase and disassembly of the MCM. The dynamics of the MCM complex is under the control of two Ser/Thr kinases, the Cell cycle-dependent kinase 2 (Cdk2) and Cell division cycle gene 7 (Cdc7). The precise substrates of the kinases at the origins and the sequence of events leading to the origins firing are not well understood. Using the two hybrid selection in yeast, we have identified a novel gene, the Cdk2 interacting protein, CINP. We show that CINP is a component of the active cyclin E /Cdk2 and cyclin A /Cdk2 complexes. CINP also interacts with Cdc7 and is phopshorylated by Cdc7, but not by Cdk2. We further show that CINP binds to chromatin in a replication-dependent manner, and associates with ORC2-containing complexes and MCM. We propose that CINP is part of the Cdc7-dependent mechanism of origin firing and a functional and physical link between Cdk2 and Cdc7 complexes at the origins.

CDK Phosphorylation of a Novel NLS-NES Module Distributed between Two Subunits of the Mcm2-7 Complex Prevents Chromosomal Rereplication.

Liku ME, Nguyen VQ, Rosales AW, Irie K, Li JJ

Mol Biol Cell :HASH(0x8bda20)
Aug 10, 2005

Monitoring Editor: Karsten Weis Cyclin-dependent kinases (CDKs) use multiple mechanisms to block reassembly of prereplicative complexes (preRCs) at replication origin to prevent inappropriate rereplication. In S. cerevisiae, one of these mechanisms promotes the net nuclear export of a preRC component, the Mcm2-7 complex, during S, G2 and M phases. Here we identify two partial nuclear localization signals (NLSs) on Mcm2 and Mcm3 that are each necessary, but not sufficient, for nuclear localization of the Mcm2-7 complex. When brought together in cis, however, the two partial signals constitute a potent NLS, sufficient for robust nuclear localization when fused to an otherwise cytoplasmic protein. We also identify a Crm1-dependent nuclear export signal (NES) adjacent to the Mcm3 NLS. Remarkably, the Mcm2-Mcm3 NLS and the Mcm3 NES are sufficient to form a transport module that recapitulates the cell cycle-regulated localization of the entire Mcm2-7 complex. Moreover, we show that CDK regulation promotes net export by phosphorylation of the Mcm3 portion of this module and that nuclear export of the Mcm2-7 complex is sufficient to disrupt replication initiation. We speculate that the distribution of partial transport signals among distinct subunits of a complex may enhance the specificity of protein localization and raise the possibility that previously undetected distributed transport signals are used by other multiprotein complexes.

The origin recognition core complex regulates dendrite and spine development in postmitotic neurons.

Huang Z, Zang K, Reichardt LF

J Cell Biol :HASH(0x8bd8d0)
Aug 8, 2005

The origin recognition complex (ORC) ensures exactly one round of genome replication per cell cycle through acting as a molecular switch that precisely controls the assembly, firing, and inactivation of the replication initiation machinery. Recent data indicate that it may also coordinate the processes of mitosis and cytokinesis and ensure the proper distribution of replicated genome to daughter cells. We have found that the ORC core subunits are highly expressed in the nervous system. They are selectively localized to the neuronal somatodendritic compartment and enriched in the membrane fraction. siRNA knockdown of ORC subunits dramatically reduced dendritic branch formation and severely impeded dendritic spine emergence. Expression of ORC ATPase motif mutants enhanced the branching of dendritic arbors. The ORC core complex thus appears to have a novel role in regulating dendrite and dendritic spine development in postmitotic neurons.

Initiation of DNA replication at the human beta-globin 3' enhancer.

Buzina A, Aladjem MI, Kolman JL, Wahl GM, Ellis J

Nucleic Acids Res 33:4412-24
, 2005

The origin of DNA replication in the human beta-globin gene contains an initiation region (IR) and two flanking auxiliary elements. Two replicator modules are located within the upstream auxiliary sequence and the IR core, but the functional sequences in the downstream auxiliary element are unknown. Here, we use a combination of benzoylated-naphthoylated DEAE (BND) cellulose purification and nascent strand abundance assays to show that replication initiation occurs at the beta-globin 3' enhancer on human chromosome 11 in the Hu11 hybrid murine erythroleukemia (MEL) cell line. To examine replicator function, 3' enhancer fragments were inserted into an ectopic site in MEL cells via an optimized FRT/EGFP-FLP integration system. These experiments demonstrate that the 1.6 kb downstream auxiliary element is a third replicator module called bGRep-E in erythroid cells. The minimal 260 bp 3' enhancer is required but not sufficient to initiate efficient replication, suggesting cooperation with adjacent sequences. The minimal 3' enhancer also cooperates with elements in an expressing HS3beta/gamma-globin construct to initiate replication. These data indicate that the beta-globin replicator has multiple initiation sites in three closely spaced replicator modules. We conclude that a mammalian enhancer can cooperate with adjacent sequences to create an efficient replicator module.

Human CDK2 Inhibition Modifies the Dynamics of Chromatin-Bound Minichromosome Maintenance Complex and Replication Protein A.

Zhu Y, Ishimi Y, Tanudji M, Lees E

Cell Cycle 4:HASH(0x8bc894)
Sep 26, 2005

Minichromosome maintenance (MCM) proteins form a complex and possess helicase activity to unwind the DNA duplex and establish a replication fork. To assure that origins only fire once per cell cycle, the MCM complex is removed from chromatin and inactivated as cells exit S phase. In this report, we demonstrate that CDK2 depletion in human cells leads to an overall phosphorylation defect at mitosis with increased rereplication, correlated with the accumulation of chromatin-bound MCM proteins. We show that CDK2 suppression results in decreased MCM4 phosphorylation at multiple serine and threonine sites. In addition, CDK2 inhibition induces an increase in chromatin-bound replication protein A (RPA) which should bind to single-stranded DNA regions, possibly establishing a replication intermediate that activates the ATR cascade. Finally, we observe that loss of CDK2 function in G(1) delays replication initiation while it promotes rereplication in G(2)/M. Thus, by modulating the phospho-status of MCM4 and regulating origin firing, S phase CDK2 appears to be an integrated component of cellular machinery required for temporally controlling replication activity and maintaining genomic stability.

Evolutionary clues to eukaryotic DNA clamp-loading mechanisms: analysis of the functional constraints imposed on replication factor C AAA+ ATPases.

Neuwald AF

Nucleic Acids Res 33:3614-28
, 2005

Ring-shaped sliding clamps encircle DNA and bind to DNA polymerase, thereby preventing it from falling off during DNA replication. In eukaryotes, sliding clamps are loaded onto DNA by the replication factor C (RFC) complex, which consists of five distinct subunits (A-E), each of which contains an AAA+ module composed of a RecA-like alpha/beta ATPase domain followed by a helical domain. AAA+ ATPases mediate chaperone-like protein remodeling. Despite remarkable progress in our understanding of clamp loaders, it is still unclear how recognition of primed DNA by RFC triggers ATP hydrolysis and how hydrolysis leads to conformational changes that can load the clamp onto DNA. While these questions can, of course, only be resolved experimentally, the design of such experiments is itself non-trivial and requires that one first formulate the right hypotheses based on preliminary observations. The functional constraints imposed on protein sequences during evolution are potential sources of information in this regard, inasmuch as these presumably are due to and thus reflect underlying mechanisms. Here, rigorous statistical procedures are used to measure and compare the constraints imposed on various RFC clamp-loader subunits, each of which performs a related but somewhat different, specialized function. Visualization of these constraints, within the context of the RFC structure, provides clues regarding clamp-loader mechanisms--suggesting, for example, that RFC-A possesses a triggering component for DNA-dependent ATP hydrolysis. It also suggests that, starting with RFC-A, four RFC subunits (A-D) are sequentially activated through a propagated switching mechanism in which a conserved arginine swings away from a position that disrupts the catalytic Walker B region and into contact with DNA thread through the center of the RFC/clamp complex. Strong constraints near regions of interaction between subunits and with the clamp likewise provide clues regarding possible coupling of hydrolysis-driven conformational changes to the clamp's release and loading onto DNA.

The promiscuous primase.

Lao-Sirieix SH, Pellegrini L, Bell SD

Trends Genet :HASH(0x7742bc)
Aug 8, 2005

DNA primases are essential for the initiation of DNA replication and progression of the replication fork. Recent phylogenetic analyses coupled with biochemical and structural studies have revealed that the arrangement of catalytic residues within the archaeal and eukaryotic primase has significant similarity to those of the Pol X family of DNA-repair polymerases. Furthermore, two additional groups of enzymes, the ligase/primase of the bacterial nonhomologous end-joining machinery and a putative replicase from an archaeal plasmid have shown striking functional and structural similarities to the core primase. The promiscuous nature of the archaeal primases suggests that these proteins might have additional roles in DNA repair in the archaea.

Identifying gene-independent noncoding functional elements in the yeast ribosomal DNA by phylogenetic footprinting.

Ganley AR, Hayashi K, Horiuchi T, Kobayashi T

Proc Natl Acad Sci U S A :HASH(0x8bddf4)
Aug 4, 2005

Sequences involved in the regulation of genetic and genomic processes are primarily located in noncoding regions. Identifying such cis-acting sequences from sequence data is difficult because their patterns are not readily apparent, and, to date, identification has concentrated on regions associated with gene-coding functions. Here, we used phylogenetic footprinting to look for gene-independent noncoding elements in the ribosomal RNA gene repeats from several Saccharomyces species. Similarity plots of ribosomal intergenic spacer alignments from six closely related Saccharomyces species allowed the identification of previously characterized functional elements, such as the origin-of-replication and replication-fork barrier sites, demonstrating that this method is a powerful predictor of noncoding functional elements. Seventeen previously uncharacterized elements, showing high levels of conservation, were also discovered. The conservation of these elements suggests that they are functional, and we demonstrate the functionality of two classes of these elements, a putative bidirectional noncoding promoter and a series of conserved peaks with matches to the origin-of-replication core consensus. Our results paint a comprehensive picture of the functionality of the Saccharomyces ribosomal intergenic region and demonstrate that functional elements not involved in gene-coding function can be identified by using comparative genomics based on sequence conservation.

Solution structure of the DNA-binding domain of RPA from Saccharomyces cerevisiae and its interaction with single-stranded DNA and SV40 T antigen.

Park CJ, Lee JH, Choi BS

Nucleic Acids Res 33:4172-81
, 2005

Replication protein A (RPA) is a three-subunit complex with multiple roles in DNA metabolism. DNA-binding domain A in the large subunit of human RPA (hRPA70A) binds to single-stranded DNA (ssDNA) and is responsible for the species-specific RPA-T antigen (T-ag) interaction required for Simian virus 40 replication. Although Saccharomyces cerevisiae RPA70A (scRPA70A) shares high sequence homology with hRPA70A, the two are not functionally equivalent. To elucidate the similarities and differences between these two homologous proteins, we determined the solution structure of scRPA70A, which closely resembled the structure of hRPA70A. The structure of ssDNA-bound scRPA70A, as simulated by residual dipolar coupling-based homology modeling, suggested that the positioning of the ssDNA is the same for scRPA70A and hRPA70A, although the conformational changes that occur in the two proteins upon ssDNA binding are not identical. NMR titrations of hRPA70A with T-ag showed that the T-ag binding surface is separate from the ssDNA-binding region and is more neutral than the corresponding part of scRPA70A. These differences might account for the species-specific nature of the hRPA70A-T-ag interaction. Our results provide insight into how these two homologous RPA proteins can exhibit functional differences, but still both retain their ability to bind ssDNA.

Regulation of histone synthesis and nucleosome assembly.

Gunjan A, Paik J, Verreault A

Biochimie 87:625-35
Jul , 2005

Histone deposition onto nascent DNA is the first step in the process of chromatin assembly during DNA replication. The process of nucleosome assembly represents a daunting task for S-phase cells, partly because cells need to rapidly package nascent DNA into nucleosomes while avoiding the generation of excess histones. Consequently, cells have evolved a number of nucleosome assembly factors and regulatory mechanisms that collectively function to coordinate the rates of histone and DNA synthesis during both normal cell cycle progression and in response to conditions that interfere with DNA replication.

Checkpoint responses to replication fork barriers.

Lambert S, Carr AM

Biochimie 87:591-602
Jul , 2005

The fidelity of DNA replication is of paramount importance to the maintenance of genome integrity. When an active replication fork is perturbed, multiple cellular pathways are recruited to stabilize the replication apparatus and to help to bypass or correct the causative problem. However, if the problem is not corrected, the fork may collapse, exposing free DNA ends to potentially inappropriate processing. In prokaryotes, replication fork collapse promotes the activity of recombination proteins to restore a replication fork. Recent work has demonstrated that recombination is also intimately linked to replication in eukaryotic cells, and that recombination proteins are recruited to collapsed, but not stalled, replication forks. In this review we discuss the different types of potential replication fork barriers (RFB) and how these distinct RFBs can result in different DNA structures at the stalled replication fork. The DNA structure checkpoints which act within S phase respond to different RFBs in different ways and we thus discuss the processes that are controlled by the DNA replication checkpoints, paying particular attention to the function of the intra-S phase checkpoint that stabilises the stalled fork.

Crosstalk between SUMO and ubiquitin on PCNA is mediated by recruitment of the helicase Srs2p.

Papouli E, Chen S, Davies AA, Huttner D, Krejci L, Sung P, Ulrich HD

Mol Cell 19:123-33
Jul 1, 2005

Posttranslational modification of proliferating cell nuclear antigen (PCNA), an essential processivity clamp for DNA polymerases, by ubiquitin and SUMO contributes to the coordination of DNA replication, damage tolerance, and mutagenesis. Whereas ubiquitination in response to DNA damage promotes the bypass of replication-blocking lesions, sumoylation during S phase is damage independent. As both modifiers target the same site on PCNA, an antagonistic action of SUMO on ubiquitin-dependent DNA damage tolerance has been proposed. We now present evidence that the apparent negative effect of SUMO on lesion bypass is not due to competition with ubiquitination but is rather mediated by the helicase Srs2p, which affects genome stability by suppressing unscheduled homologous recombination. We show that Srs2p physically interacts with sumoylated PCNA, which contributes to the recruitment of the helicase to replication forks. Our findings suggest a mechanism by which SUMO and ubiquitin cooperatively control the choice of pathway for the processing of DNA lesions during replication.

Cyclin-dependent kinase (CDK) phosphorylation destabilizes somatic Wee1 via multiple pathways.

Watanabe N, Arai H, Iwasaki JI, Shiina M, Ogata K, Hunter T, Osada H

Proc Natl Acad Sci U S A :HASH(0x7764bc)
Aug 5, 2005

At the onset of M phase, the activity of somatic Wee1 (Wee1A), the inhibitory kinase for cyclin-dependent kinase (CDK), is down-regulated primarily through proteasome-dependent degradation after ubiquitination by the E3 ubiquitin ligase SCF(beta-TrCP). The F-box protein beta-TrCP (beta-transducin repeat-containing protein), the substrate recognition component of the ubiquitin ligase, binds to its substrates through a conserved binding motif (phosphodegron) containing two phosphoserines, DpSGXXpS. Although Wee1A lacks this motif, phosphorylation of serines 53 and 123 (S53 and S123) of Wee1A by polo-like kinase 1 (Plk1) and CDK, respectively, are required for binding to beta-TrCP. The sequence surrounding phosphorylated S53 (DpSAFQE) is similar to the conserved beta-TrCP-binding motif; however, the role of S123 phosphorylation (EEGFGSSpSPVK) in beta-TrCP binding was not elucidated. In the present study, we show that phosphorylation of S123 (pS123) by CDK promoted the binding of Wee1A to beta-TrCP through three independent mechanisms. The pS123 not only directly interacted with basic residues in the WD40 repeat domain of beta-TrCP but also primed phosphorylation by two independent protein kinases, Plk1 and CK2 (formerly casein kinase 2), to create two phosphodegrons on Wee1A. In the case of Plk1, S123 phosphorylation created a polo box domain-binding motif (SpSP) on Wee1A to accelerate phosphorylation of S53 by Plk1. CK2 could phosphorylate S121, but only if S123 was phosphorylated first, thereby generating the second beta-TrCP-binding site (EEGFGpS121). Using a specific inhibitor of CK2, we showed that the phosphorylation-dependent degradation of Wee1A is important for the proper onset of mitosis.

Cdk1-dependent regulation of the mitotic inhibitor wee1.

Harvey SL, Charlet A, Haas W, Gygi SP, Kellogg DR

Cell 122:407-20
Aug 12, 2005

The Wee1 kinase phosphorylates and inhibits cyclin-dependent kinase 1 (Cdk1), thereby delaying entry into mitosis until appropriate conditions have been met. An understanding of the mechanisms that regulate Wee1 should provide new insight into how cells make the decision to enter mitosis. We report here that Swe1, the budding-yeast homolog of Wee1, is directly regulated by Cdk1. Phosphorylation of Swe1 by Cdk1 activates Swe1 and is required for formation of a stable Swe1-Cdk1 complex that maintains Cdk1 in the inhibited state. Dephosphorylation of Cdk1 leads to further phosphorylation of Swe1 and release of Cdk1. Thus, Cdk1 both positively and negatively regulates its own inhibitor. Regulation of the Swe1-Cdk1 complex is likely to play a critical role in controlling the transition into mitosis.

A high-resolution map of active promoters in the human genome.

Kim TH, Barrera LO, Zheng M, Qu C, Singer MA, Richmond TA, Wu Y, Green RD, Ren B

Nature 436:876-80
Aug 11, 2005

In eukaryotic cells, transcription of every protein-coding gene begins with the assembly of an RNA polymerase II preinitiation complex (PIC) on the promoter. The promoters, in conjunction with enhancers, silencers and insulators, define the combinatorial codes that specify gene expression patterns. Our ability to analyse the control logic encoded in the human genome is currently limited by a lack of accurate information regarding the promoters for most genes. Here we describe a genome-wide map of active promoters in human fibroblast cells, determined by experimentally locating the sites of PIC binding throughout the human genome. This map defines 10,567 active promoters corresponding to 6,763 known genes and at least 1,196 un-annotated transcriptional units. Features of the map suggest extensive use of multiple promoters by the human genes and widespread clustering of active promoters in the genome. In addition, examination of the genome-wide expression profile reveals four general classes of promoters that define the transcriptome of the cell. These results provide a global view of the functional relationships among transcriptional machinery, chromatin structure and gene expression in human cells.