RESUMO
To initiate DNA replication, it is essential to properly assemble a pair of replicative helicases at each replication origin. While the general principle of this process applies universally from prokaryotes to eukaryotes, the specific mechanisms governing origin selection, helicase loading, and subsequent helicase activation vary significantly across different species. Recent advancements in cryo-electron microscopy (cryo-EM) have revolutionized our ability to visualize large protein or protein-DNA complexes involved in the initiation of DNA replication. Complemented by real-time single-molecule analysis, the available high-resolution cryo-EM structures have greatly enhanced our understanding of the dynamic regulation of this process at origin DNA. This review primarily focuses on the latest structural discoveries that shed light on the key molecular machineries responsible for driving replication initiation, with a particular emphasis on the assembly of pre-replication complex (pre-RC) in eukaryotes.
RESUMO
In eukaryotes, DNA compacts into chromatin through nucleosomes1,2. Replication of the eukaryotic genome must be coupled to the transmission of the epigenome encoded in the chromatin3,4. Here we report cryo-electron microscopy structures of yeast (Saccharomyces cerevisiae) replisomes associated with the FACT (facilitates chromatin transactions) complex (comprising Spt16 and Pob3) and an evicted histone hexamer. In these structures, FACT is positioned at the front end of the replisome by engaging with the parental DNA duplex to capture the histones through the middle domain and the acidic carboxyl-terminal domain of Spt16. The H2A-H2B dimer chaperoned by the carboxyl-terminal domain of Spt16 is stably tethered to the H3-H4 tetramer, while the vacant H2A-H2B site is occupied by the histone-binding domain of Mcm2. The Mcm2 histone-binding domain wraps around the DNA-binding surface of one H3-H4 dimer and extends across the tetramerization interface of the H3-H4 tetramer to the binding site of Spt16 middle domain before becoming disordered. This arrangement leaves the remaining DNA-binding surface of the other H3-H4 dimer exposed to additional interactions for further processing. The Mcm2 histone-binding domain and its downstream linker region are nested on top of Tof1, relocating the parental histones to the replisome front for transfer to the newly synthesized lagging-strand DNA. Our findings offer crucial structural insights into the mechanism of replication-coupled histone recycling for maintaining epigenetic inheritance.
Assuntos
Cromatina , Replicação do DNA , Epistasia Genética , Histonas , Saccharomyces cerevisiae , Sítios de Ligação , Cromatina/química , Cromatina/genética , Cromatina/metabolismo , Cromatina/ultraestrutura , Microscopia Crioeletrônica , Replicação do DNA/genética , DNA Fúngico/biossíntese , DNA Fúngico/química , DNA Fúngico/metabolismo , DNA Fúngico/ultraestrutura , Epistasia Genética/genética , Histonas/química , Histonas/metabolismo , Histonas/ultraestrutura , Complexos Multienzimáticos/química , Complexos Multienzimáticos/metabolismo , Complexos Multienzimáticos/ultraestrutura , Nucleossomos/química , Nucleossomos/metabolismo , Nucleossomos/ultraestrutura , Ligação Proteica , Domínios Proteicos , Multimerização Proteica , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/ultraestrutura , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/ultraestruturaRESUMO
The replisome that replicates the eukaryotic genome consists of at least three engines: the Cdc45-MCM-GINS (CMG) helicase that separates duplex DNA at the replication fork and two DNA polymerases, one on each strand, that replicate the unwound DNA. Here, we determined a series of cryo-electron microscopy structures of a yeast replisome comprising CMG, leading-strand polymerase Polε and three accessory factors on a forked DNA. In these structures, Polε engages or disengages with the motor domains of the CMG by occupying two alternative positions, which closely correlate with the rotational movement of the single-stranded DNA around the MCM pore. During this process, the polymerase remains stably coupled to the helicase using Psf1 as a hinge. This synergism is modulated by a concerted rearrangement of ATPase sites to drive DNA translocation. The Polε-MCM coupling is not only required for CMG formation to initiate DNA replication but also facilitates the leading-strand DNA synthesis mediated by Polε. Our study elucidates a mechanism intrinsic to the replisome that coordinates the activities of CMG and Polε to negotiate any roadblocks, DNA damage, and epigenetic marks encountered during translocation along replication forks.
Assuntos
DNA Helicases , DNA Polimerase Dirigida por DNA , Microscopia Crioeletrônica , DNA Helicases/genética , Replicação do DNA , Saccharomyces cerevisiae/genéticaRESUMO
Methylation of histone H3 lysine-79 (H3K79) is an epigenetic mark for gene regulation in development, cellular differentiation, and disease progression. However, how this histone mark is translated into downstream effects remains poorly understood owing to a lack of knowledge about its readers. We developed a nucleosome-based photoaffinity probe to capture proteins that recognize H3K79 dimethylation (H3K79me2) in a nucleosomal context. In combination with a quantitative proteomics approach, this probe identified menin as a H3K79me2 reader. A cryo-electron microscopy structure of menin bound to an H3K79me2 nucleosome revealed that menin engages with the nucleosome using its fingers and palm domains and recognizes the methylation mark through a π-cation interaction. In cells, menin is selectively associated with H3K79me2 on chromatin, particularly in gene bodies.
Assuntos
Epigênese Genética , Histonas , Lisina , Nucleossomos , Proteínas Proto-Oncogênicas , Cromatina/metabolismo , Microscopia Crioeletrônica , Histonas/química , Histonas/metabolismo , Metilação , Nucleossomos/química , Nucleossomos/metabolismo , Lisina/metabolismo , Proteômica/métodos , Proteínas Proto-Oncogênicas/metabolismo , Humanos , Animais , Sondas Moleculares/química , Processamento de Proteína Pós-TraducionalRESUMO
In eukaryotes, DNA replication initiation requires assembly and activation of the minichromosome maintenance (MCM) 2-7 double hexamer (DH) to melt origin DNA strands. However, the mechanism for this initial melting is unknown. Here, we report a 2.59-Å cryo-electron microscopy structure of the human MCM-DH (hMCM-DH), also known as the pre-replication complex. In this structure, the hMCM-DH with a constricted central channel untwists and stretches the DNA strands such that almost a half turn of the bound duplex DNA is distorted with 1 base pair completely separated, generating an initial open structure (IOS) at the hexamer junction. Disturbing the IOS inhibits DH formation and replication initiation. Mapping of hMCM-DH footprints indicates that IOSs are distributed across the genome in large clusters aligning well with initiation zones designed for stochastic origin firing. This work unravels an intrinsic mechanism that couples DH formation with initial DNA melting to license replication initiation in human cells.
Assuntos
Replicação do DNA , Humanos , Proteínas de Ciclo Celular/metabolismo , Microscopia Crioeletrônica , Proteínas de Ligação a DNA/metabolismo , Proteínas de Manutenção de Minicromossomo/metabolismo , Origem de ReplicaçãoRESUMO
DNA replication initiation in eukaryotes is tightly regulated through two cell-cycle specific processes, replication licensing to install inactive minichromosome maintenance (MCM) double-hexamers (DH) on origins in early G1 phase and origin firing to assemble and activate Cdc45-Mcm2-7-GINS (CMG) helicases upon S phase entry. Two kinases, cyclin-dependent kinase (CDK) and Dbf4-dependent kinase (DDK), are responsible for driving the association of replication factors with the MCM-DH to form CMG helicases for origin melting and DNA unwinding and eventually replisomes for bi-directional DNA synthesis. In recent years, cryo-electron microscopy studies have generated a collection of structural snapshots for the stepwise assembly and remodeling of the replication initiation machineries, creating a framework for understanding the regulation of this fundamental process at a molecular level. Very recent progress is the structural characterization of the elusive MCM-DH-DDK complex, which provides insights into mechanisms of kinase activation, substrate recognition and selection, as well as molecular role of DDK-mediated MCM-DH phosphorylation in helicase activation.
Assuntos
Proteínas Serina-Treonina Quinases , Proteínas de Saccharomyces cerevisiae , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Microscopia Crioeletrônica , Proteínas de Ligação a DNA/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Replicação do DNA , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Proteínas de Manutenção de Minicromossomo/química , Proteínas de Manutenção de Minicromossomo/genética , Proteínas de Manutenção de Minicromossomo/metabolismo , DNA , Origem de ReplicaçãoRESUMO
Understanding human DNA replication through the study of yeast has been an extremely fruitful journey. The minichromosome maintenance (MCM) 2-7 genes that encode the catalytic core of the eukaryotic replisome were initially identified through forward yeast genetics. The origin recognition complexes (ORC) that load the MCM hexamers at replication origins were purified from yeast extracts. We have reached an age where high-resolution cryoEM structures of yeast and human replication complexes can be compared side-by-side. Their similarities and differences are converging as alternative strategies that may deviate in detail but are shared by both species.
RESUMO
The Dbf4-dependent kinase Cdc7 (DDK) regulates DNA replication initiation by phosphorylation of the MCM double hexamer (MCM-DH) to promote helicase activation. Here, we determine a series of cryo electron microscopy (cryo-EM) structures of yeast DDK bound to the MCM-DH. These structures, occupied by one or two DDKs, differ primarily in the conformations of the kinase core. The interactions of DDK with the MCM-DH are mediated exclusively by subunit Dbf4 straddling across the hexamer interface on the three N-terminal domains (NTDs) of subunits Mcm2, Mcm6, and Mcm4. This arrangement brings Cdc7 close to its only essential substrate, the N-terminal serine/threonine-rich domain (NSD) of Mcm4. Dbf4 further displaces the NSD from its binding site on Mcm4-NTD, facilitating an immediate targeting of this motif by Cdc7. Moreover, the active center of Cdc7 is occupied by a unique Dbf4 inhibitory loop, which is disengaged when the kinase core assumes wobbling conformations. This study elucidates the versatility of Dbf4 in regulating the ordered multisite phosphorylation of the MCM-DH by Cdc7 kinase during helicase activation.
Assuntos
Proteínas de Saccharomyces cerevisiae , Proteínas de Ciclo Celular/metabolismo , Replicação do DNA , Componente 6 do Complexo de Manutenção de Minicromossomo/metabolismo , Fosforilação , Proteínas Serina-Treonina Quinases , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMO
The Origin Recognition Complex (ORC) is an evolutionarily conserved six-subunit protein complex that binds specific sites at many locations to coordinately replicate the entire eukaryote genome. Though highly conserved in structure, ORC's selectivity for replication origins has diverged tremendously between yeasts and humans to adapt to vastly different life cycles. In this work, we demonstrate that the selectivity determinant of ORC for DNA binding lies in a 19-amino acid insertion helix in the Orc4 subunit, which is present in yeast but absent in human. Removal of this motif from Orc4 transforms the yeast ORC, which selects origins based on base-specific binding at defined locations, into one whose selectivity is dictated by chromatin landscape and afforded with plasticity, as reported for human. Notably, the altered yeast ORC has acquired an affinity for regions near transcriptional start sites (TSSs), which the human ORC also favors.
Assuntos
Complexo de Reconhecimento de Origem/metabolismo , Saccharomyces cerevisiae/metabolismo , Sequência de Aminoácidos , Sequência de Bases , Sítios de Ligação , DNA Fúngico/metabolismo , Fase G2/genética , Genoma Fúngico , Humanos , Modelos Genéticos , Mutação/genética , Nucleossomos/metabolismo , Motivos de Nucleotídeos/genética , Complexo de Reconhecimento de Origem/química , Fase S , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Processos Estocásticos , Sítio de Iniciação de TranscriçãoRESUMO
The function of the origin recognition complex (ORC) in DNA replication is highly conserved in recognizing and marking the initiation sites. The detailed molecular mechanisms by which human ORC is reconfigured into a state competent for origin association remain largely unknown. Here, we present structural characterizations of human ORC1-5 and ORC2-5 assemblies. ORC2-5 exhibits a tightly autoinhibited conformation with the winged-helix domain of ORC2 completely blocking the central DNA-binding channel. The binding of ORC1 partially relieves the autoinhibitory effect of ORC2-5 through remodeling ORC2-WHD, which makes ORC2-WHD away from the central channel creating a still autoinhibited but more dynamic structure. In particular, the AAA+ domain of ORC1 is highly flexible to sample a variety of conformations from inactive to potentially active states. These results provide insights into the detailed mechanisms regulating the autoinhibition of human ORC and its subsequent activation for DNA binding.
RESUMO
The six-subunit origin recognition complex (ORC) binds to DNA to mark the site for the initiation of replication in eukaryotes. Here we report a 3 Å cryo-electron microscopy structure of the Saccharomyces cerevisiae ORC bound to a 72-base-pair origin DNA sequence that contains the ARS consensus sequence (ACS) and the B1 element. The ORC encircles DNA through extensive interactions with both phosphate backbone and bases, and bends DNA at the ACS and B1 sites. Specific recognition of thymine residues in the ACS is carried out by a conserved basic amino acid motif of Orc1 in the minor groove, and by a species-specific helical insertion motif of Orc4 in the major groove. Moreover, similar insertions into major and minor grooves are also embedded in the B1 site by basic patch motifs from Orc2 and Orc5, respectively, to contact bases and to bend DNA. This work pinpoints a conserved role of ORC in modulating DNA structure to facilitate origin selection and helicase loading in eukaryotes.
Assuntos
Microscopia Crioeletrônica , Complexo de Reconhecimento de Origem/química , Complexo de Reconhecimento de Origem/ultraestrutura , Origem de Replicação , Saccharomyces cerevisiae , Sequência de Bases , DNA/química , DNA/genética , DNA/metabolismo , DNA/ultraestrutura , Proteínas de Manutenção de Minicromossomo/metabolismo , Modelos Moleculares , Complexo de Reconhecimento de Origem/metabolismo , Subunidades Proteicas/química , Subunidades Proteicas/metabolismo , Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/ultraestrutura , Especificidade por SubstratoRESUMO
A family of six homologous subunits, Mcm2, -3, -4, -5, -6, and -7, each with its own unique features, forms the catalytic core of the eukaryotic replicative helicase. The necessity of six similar but non-identical subunits has been a mystery since its initial discovery. Recent cryo-EM structures of the Mcm2-7 (MCM) double hexamer, its precursors, and the origin recognition complex (ORC)-Cdc6-Cdt1-Mcm2-7 (OCCM) intermediate showed that each of these subunits plays a distinct role in orchestrating the assembly of the pre-replication complex (pre-RC) by ORC-Cdc6 and Cdt1.
Assuntos
Replicação do DNA , Proteínas de Manutenção de Minicromossomo/metabolismo , Complexo de Reconhecimento de Origem/metabolismo , Animais , Domínio Catalítico , Proteínas de Ciclo Celular/metabolismo , Humanos , Proteínas de Manutenção de Minicromossomo/química , Proteínas de Manutenção de Minicromossomo/ultraestrutura , Modelos Moleculares , Complexos Multiproteicos , Proteínas Nucleares/metabolismo , Conformação de Ácido Nucleico , Complexo de Reconhecimento de Origem/química , Complexo de Reconhecimento de Origem/ultraestrutura , Ligação Proteica , Subunidades Proteicas , Relação Estrutura-AtividadeRESUMO
The minichromosome maintenance complex (MCM) hexameric complex (Mcm2-7) forms the core of the eukaryotic replicative helicase. During G1 phase, two Cdt1-Mcm2-7 heptamers are loaded onto each replication origin by the origin-recognition complex (ORC) and Cdc6 to form an inactive MCM double hexamer (DH), but the detailed loading mechanism remains unclear. Here we examine the structures of the yeast MCM hexamer and Cdt1-MCM heptamer from Saccharomyces cerevisiae. Both complexes form left-handed coil structures with a 10-15-Å gap between Mcm5 and Mcm2, and a central channel that is occluded by the C-terminal domain winged-helix motif of Mcm5. Cdt1 wraps around the N-terminal regions of Mcm2, Mcm6 and Mcm4 to stabilize the whole complex. The intrinsic coiled structures of the precursors provide insights into the DH formation, and suggest a spring-action model for the MCM during the initial origin melting and the subsequent DNA unwinding.
Assuntos
Proteínas de Ciclo Celular/química , Proteínas de Ligação a DNA/química , Proteínas de Manutenção de Minicromossomo/química , Multimerização Proteica , Proteínas de Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/metabolismo , Adenilil Imidodifosfato/química , Motivos de Aminoácidos , Proteínas de Ciclo Celular/metabolismo , Proteínas de Ciclo Celular/ultraestrutura , Microscopia Crioeletrônica , Proteínas de Ligação a DNA/metabolismo , Proteínas de Ligação a DNA/ultraestrutura , Proteínas de Manutenção de Minicromossomo/metabolismo , Proteínas de Manutenção de Minicromossomo/ultraestrutura , Modelos Moleculares , Domínios Proteicos , Estrutura Secundária de Proteína , Subunidades Proteicas/química , Subunidades Proteicas/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/ultraestrutura , Dedos de ZincoRESUMO
The eukaryotic minichromosome maintenance 2-7 complex is the core of the inactive MCM replication licensing complex and the catalytic core of the Cdc45-MCM-GINS replicative helicase. The years of effort to determine the structure of parts or the whole of the heterohexameric complex by X-ray crystallography and conventional cryo-EM produced limited success. Modern cryo-EM technology ushered in a new era of structural biology that allowed the determination of the structure of the inactive double hexamer at an unprecedented resolution of 3.8 Å. This review will focus on the fine details observed in the Mcm2-7 double hexameric complex and their implications for the function of the Mcm2-7 hexamer in its different roles during DNA replication.
Assuntos
Replicação do DNA/fisiologia , Proteínas de Manutenção de Minicromossomo/química , Proteínas de Manutenção de Minicromossomo/fisiologia , Multimerização Proteica , Estrutura Quaternária de Proteína/fisiologia , Animais , Domínio Catalítico , HumanosRESUMO
DNA replication in eukaryotes is strictly regulated by several mechanisms. A central step in this replication is the assembly of the heterohexameric minichromosome maintenance (MCM2-7) helicase complex at replication origins during G1 phase as an inactive double hexamer. Here, using cryo-electron microscopy, we report a near-atomic structure of the MCM2-7 double hexamer purified from yeast G1 chromatin. Our structure shows that two single hexamers, arranged in a tilted and twisted fashion through interdigitated amino-terminal domain interactions, form a kinked central channel. Four constricted rings consisting of conserved interior ß-hairpins from the two single hexamers create a narrow passageway that tightly fits duplex DNA. This narrow passageway, reinforced by the offset of the two single hexamers at the double hexamer interface, is flanked by two pairs of gate-forming subunits, MCM2 and MCM5. These unusual features of the twisted and tilted single hexamers suggest a concerted mechanism for the melting of origin DNA that requires structural deformation of the intervening DNA.
Assuntos
Microscopia Crioeletrônica , Proteínas de Manutenção de Minicromossomo/química , Proteínas de Manutenção de Minicromossomo/ultraestrutura , Subunidades Proteicas/química , Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/ultraestrutura , Sítios de Ligação , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/metabolismo , Proteínas de Ciclo Celular/ultraestrutura , Cromatina/química , Sequência Conservada , DNA/química , DNA/metabolismo , DNA/ultraestrutura , DNA Polimerase Dirigida por DNA/química , DNA Polimerase Dirigida por DNA/ultraestrutura , Fase G1 , Proteínas de Manutenção de Minicromossomo/metabolismo , Modelos Biológicos , Modelos Moleculares , Complexos Multienzimáticos/química , Complexos Multienzimáticos/ultraestrutura , Desnaturação de Ácido Nucleico , Ligação Proteica , Multimerização Proteica , Estrutura Terciária de Proteína , Subunidades Proteicas/metabolismo , Origem de Replicação , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/ultraestruturaRESUMO
We report a robust two-color method for super-resolution localization microscopy. Two-dye combination of Alexa647 and Alexa750 in an imaging buffer containing COT and using TCEP as switching regent provides matched and balanced switching characteristics for both dyes, allowing simultaneous capture of both on a single camera. Active sample locking stabilizes sample with 1nm accuracy during imaging. With over 4,000 photons emitted from both dyes, two-color superresolution images with high-quality were obtained in a wide range of samples including cell cultures, tissue sections and yeast cells.
RESUMO
Several replication-initiation proteins are assembled stepwise onto replicators to form pre-replicative complexes (pre-RCs) to license eukaryotic DNA replication. We performed a yeast functional proteomic screen and identified the Rix1 complex members (Ipi1p-Ipi2p/Rix1-Ipi3p) as pre-RC components and critical determinants of replication licensing and replication-initiation frequency. Ipi3p interacts with pre-RC proteins, binds chromatin predominantly at ARS sequences in a cell cycle-regulated and ORC- and Noc3p-dependent manner and is required for loading Cdc6p, Cdt1p and MCM onto chromatin to form pre-RC during the M-to-G1 transition and for pre-RC maintenance in G1 phase-independent of its role in ribosome biogenesis. Moreover, Ipi1p and Ipi2p, but not other ribosome biogenesis proteins Rea1p and Utp1p, are also required for pre-RC formation and maintenance, and Ipi1p, -2p and -3p are interdependent for their chromatin association and function in pre-RC formation. These results establish a new framework for the hierarchy of pre-RC proteins, where the Ipi1p-2p-3p complex provides a critical link between ORC-Noc3p and Cdc6p-Cdt1p-MCM in replication licensing.
Assuntos
Replicação do DNA , Ribossomos/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Ciclo Celular/genética , Ciclo Celular/fisiologia , Proteínas de Ciclo Celular/metabolismo , Cromatina/metabolismo , DNA Helicases/genética , DNA Helicases/metabolismo , Proteínas de Ligação a DNA/metabolismo , Fase G1/fisiologia , Proteína 1 de Manutenção de Minicromossomo/metabolismo , Proteínas Nucleares/metabolismo , Proteínas de Transporte Nucleocitoplasmático/metabolismo , Complexo de Reconhecimento de Origem/genética , Complexo de Reconhecimento de Origem/metabolismo , Proteoma , Origem de Replicação , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Transativadores/genética , Transativadores/metabolismoRESUMO
In eukaryotes, replication licensing is achieved through sequential loading of several replication-initiation proteins onto replication origins to form pre-replicative complexes (pre-RCs), and unscheduled replication licensing is prevented by cyclin-dependent kinases (CDKs) through inhibitory phosphorylations of multiple initiation proteins. It is known that CDK inactivation during mitotic exit promotes pre-RC formation for the next cell cycle. However, whether the removal of the inhibitory phosphorylations on the initiation proteins is essential and the identity of the acting phosphatase(s) remain unknown. Here, we show that cell division cycle protein 14 (Cdc14p) dephosphorylates replication-initiation proteins Orc2p, Orc6p, Cdc6p and Mcm3p to restore their competence for pre-RC assembly in the budding yeast Saccharomyces cerevisiae. Cells without functional Cdc14p fail to dephosphorylate initiation proteins and to form pre-RCs - even when CDK activities are suppressed - and cannot replicate DNA in mitotic rereplication systems, whereas pulsed ectopic expression of Cdc14p in mitotic cells results in efficient pre-RC assembly and DNA rereplication. Furthermore, Cdc14p becomes dispensable for DNA rereplication in mitotic cells with combined non-phosphorylatable and/or phosphorylation-insensitive alleles of the initiation proteins. These data unravel the essential role of Cdc14p in replication licensing, beyond its established functions in mitotic exit, providing new insight into the intricate regulation of DNA replication through the interplay of CDKs and the Cdc14p phosphatase.