S-phase Cyclin-dependent Kinase Research Articles

S-CDK-regulated bipartite interaction of Mcm10 with MCM is essential for DNA replication

Mcm10 plays an essential role in the activation of replicative helicase CMG through the cell cycle-regulated interaction with the prototype MCM double hexamer in Saccharomyces cerevisiae. In this study, we reported that Mcm10 is phosphorylated by S-phase cyclin-dependent kinases (S-CDKs) at S66, which enhances Mcm10–-MCM association during the S phase. S66A single mutation or even deletion of whole N-terminus (a.a. 1–128) only causes mild growth defects. Nevertheless, S66 becomes indispensable in the absence of the Mcm10 C-terminus ((a.a. 463–571), the major MCM-binding domain. Using a two-degron strategy to efficiently deplete Mcm10, we show that mcm10-S66AΔC has a severe defect in proceeding into the S phase. Notably, both lethality and S-phase deficiency can be rescued by artificially tethering mcm10-S66AΔC to MCM. These findings illustrate how the Mcm10–MCM association is regulated as a crucial event in DNA replication initiation.

The CRK2-CYC13 complex functions as an S-phase cyclin-dependent kinase to promote DNA replication in Trypanosoma brucei

BackgroundFaithful DNA replication is essential to maintain genomic stability in all living organisms, and the regulatory pathway for DNA replication initiation is conserved from yeast to humans. The evolutionarily ancient human parasite Trypanosoma brucei, however, lacks many of the conserved DNA replication factors and may employ unusual mechanisms for DNA replication. Neither the S-phase cyclin-dependent kinase (CDK) nor the regulatory pathway governing DNA replication has been previously identified in T. brucei.ResultsHere we report that CRK2 (Cdc2-related kinase 2) complexes with CYC13 (Cyclin13) and functions as an S-phase CDK to promote DNA replication in T. brucei. We further show that CRK2 phosphorylates Mcm3, a subunit of the Mcm2–7 sub-complex of the Cdc45-Mcm2–7-GINS complex, and demonstrate that Mcm3 phosphorylation by CRK2 facilitates interaction with Sld5, a subunit of the GINS sub-complex of the Cdc45-Mcm2–7-GINS complex.ConclusionsThese results identify the CRK2-CYC13 complex as an S-phase regulator in T. brucei and reveal its role in regulating DNA replication through promoting the assembly of the Cdc45-Mcm2–7-GINS complex.

A new linear cyclin docking motif that mediates exclusively S-phase CDK-specific signaling.

Cyclin‐dependent kinases (CDKs), the master regulators of cell division, are activated by different cyclins at different cell cycle stages. In addition to being activators of CDKs, cyclins recognize various linear motifs to target CDK activity to specific proteins. We uncovered a cyclin docking motif, NLxxxL, that contributes to phosphorylation‐dependent degradation of the CDK inhibitor Far1 at the G1/S stage in the yeast Saccharomyces cerevisiae. This motif is recognized exclusively by S‐phase CDK (S‐CDK) Clb5/6‐Cdc28 and is considerably more potent than the conventional RxL docking motif. The NLxxxL and RxL motifs were found to overlap in some target proteins, suggesting that cyclin docking motifs can evolve to switch from one to another for fine‐tuning of cell cycle events. Using time‐lapse fluorescence microscopy, we show how different docking connections temporally control phosphorylation‐driven target degradation. This also revealed a differential function of the phosphoadaptor protein Cks1, as Cks1 docking potentiated degron phosphorylation of RxL‐containing but not of NLxxxL‐containing substrates. The NLxxxL motif was found to govern S‐cyclin‐specificity in multiple yeast CDK targets including Fin1, Lif1, and Slx4, suggesting its wider importance.

Molecular mechanisms of highly pathogenic viruses' replication and their applications for a novel drug discovery

Productive (lytic) replication of DNA viruses elicits host cell DNA damage responses, which cause both beneficial and detrimental effects on viral replication. Viruses utilize them and selectively cancel the 'noisy' downstream signaling pathways, leading to maintain high S-phase CDK activities required for viral replication. To achieve this fine tuning of cellular environment, herpesviruses encode many (>70) genes in their genome, which are expressed in a strictly regulated temporal cascade (immediate-early, early, and late). Here, I introduce and discuss how Epstein-Barr virus, an oncogenic herpesvirus, hijacks the cellular environment and adapt it for the progeny production.

Dynamic changes of cellular environment during Epstein-Barr virus productive replication

Productive (lytic) replication of DNA viruses elicits host cell DNA damage responses, which cause both beneficial and detrimental effects on viral replication. Viruses utilize them and selectively cancel the 'noisy' downstream signaling pathways, leading to maintain high S-phase CDK activities required for viral replication. To achieve this fine tuning of cellular environment, herpesviruses encode many (>70) genes in their genome, which are expressed in a strictly regulated temporal cascade (immediate-early, early, and late). Here, I introduce and discuss how Epstein-Barr virus, an oncogenic herpesvirus, hijacks the cellular environment and adapt it for the progeny production.

P21 limits S phase DNA damage caused by the Wee1 inhibitor MK1775

ABSTRACTThe Wee1 inhibitor MK1775 (AZD1775) is currently being tested in clinical trials for cancer treatment. Here, we show that the p53 target and CDK inhibitor p21 protects against MK1775-induced DNA damage during S-phase. Cancer and normal cells deficient for p21 (HCT116 p21-/-, RPE p21-/-, and U2OS transfected with p21 siRNA) showed higher induction of the DNA damage marker γH2AX in S-phase in response to MK1775 compared to the respective parental cells. Furthermore, upon MK1775 treatment the levels of phospho-DNA PKcs S2056 and phospho-RPA S4/S8 were higher in the p21 deficient cells, consistent with increased DNA breakage. Cell cycle analysis revealed that these effects were due to an S-phase function of p21, but MK1775-induced S-phase CDK activity was not altered as measured by CDK-dependent phosphorylations. In the p21 deficient cancer cells MK1775-induced cell death was also increased. Moreover, p21 deficiency sensitized to combined treatment of MK1775 and the CHK1-inhibitor AZD6772, and to the combination of MK1775 with ionizing radiation. These results show that p21 protects cancer cells against Wee1 inhibition and suggest that S-phase functions of p21 contribute to mediate such protection. As p21 can be epigenetically downregulated in human cancer, we propose that p21 levels may be considered during future applications of Wee1 inhibitors.

A Positive Amplification Mechanism Involving a Kinase and Replication Initiation Factor Helps Assemble the Replication Fork Helicase

The assembly of the replication fork helicase during S phase is key to the initiation of DNA replication in eukaryotic cells. One step in this assembly in budding yeast is the association of Cdc45 with the Mcm2-7 heterohexameric ATPase, and a second step is the assembly of the tetrameric GINS (GG-Ichi-Nii-San) complex with Mcm2-7. Dbf4-dependent kinase (DDK) and S-phase cyclin-dependent kinase (S-CDK) are two S phase-specific kinases that phosphorylate replication proteins during S phase, and Dpb11, Sld2, Sld3, Pol ϵ, and Mcm10 are factors that are also required for replication initiation. However, the exact roles of these initiation factors in assembly of the replication fork helicase remain unclear. We show here that Dpb11 stimulates DDK phosphorylation of the minichromosome maintenance complex protein Mcm4 alone and also of the Mcm2-7 complex and the dsDNA-loaded Mcm2-7 complex. We further demonstrate that Dpb11 can directly recruit DDK to Mcm4. A DDK phosphomimetic mutant of Mcm4 bound Dpb11 with substantially higher affinity than wild-type Mcm4, suggesting a mechanism to recruit Dpb11 to DDK-phosphorylated Mcm2-7. Furthermore, dsDNA-loaded Mcm2-7 harboring the DDK phosphomimetic Mcm4 mutant bound GINS in the presence of Dpb11, suggesting a mechanism for how GINS is recruited to Mcm2-7. We isolated a mutant of Dpb11 that is specifically defective for binding to Mcm4. This mutant, when expressed in budding yeast, diminished cell growth and DNA replication, substantially decreased Mcm4 phosphorylation, and decreased association of GINS with replication origins. We conclude that Dpb11 functions with DDK and Mcm4 in a positive amplification mechanism to trigger the assembly of the replication fork helicase.

Cyclin-dependent kinase regulates the length of S phase through TICRR/TRESLIN phosphorylation.

S-phase cyclin-dependent kinases (CDKs) stimulate replication initiation and accelerate progression through the replication timing program, but it is unknown which CDK substrates are responsible for these effects. CDK phosphorylation of the replication factor TICRR (TopBP1-interacting checkpoint and replication regulator)/TRESLIN is required for DNA replication. We show here that phosphorylated TICRR is limiting for S-phase progression. Overexpression of a TICRR mutant with phosphomimetic mutations at two key CDK-phosphorylated residues (TICRR(TESE)) stimulates DNA synthesis and shortens S phase by increasing replication initiation. This effect requires the TICRR region that is necessary for its interaction with MDM two-binding protein. Expression of TICRR(TESE) does not grossly alter the spatial organization of replication forks in the nucleus but does increase replication clusters and the number of replication forks within each cluster. In contrast to CDK hyperactivation, the acceleration of S-phase progression by TICRR(TESE) does not induce DNA damage. These results show that CDK can stimulate initiation and compress the replication timing program by phosphorylating a single protein, suggesting a simple mechanism by which S-phase length is controlled.

Coordination of Chromatid Separation and Spindle Elongation by Antagonistic Activities of Mitotic and S-Phase CDKs

Because cohesion prevents sister-chromatid separation and spindle elongation, cohesion dissolution may trigger these two events simultaneously. However, the relatively normal spindle elongation kinetics in yeast cohesin mutants indicates an additional mechanism for the temporal control of spindle elongation. Here we show evidence indicating that S-phase CDK (cyclin dependent kinase) negatively regulates spindle elongation. In contrast, mitotic CDK promotes spindle elongation by activating Cdc14 phosphatase, which reverses the protein phosphorylation imposed by S-phase CDK. Our data suggest that S-phase CDK negatively regulates spindle elongation partly through its phosphorylation of a spindle pole body (SPB) protein Spc110. We also show that hyperactive S-phase CDK compromises the microtubule localization of Stu2, a processive microtubule polymerase essential for spindle elongation. Strikingly, we found that hyperactive mitotic CDK induces uncoupled spindle elongation and sister-chromatid separation in securin mutants (pds1Δ), and we speculate that asynchronous chromosome segregation in pds1Δ cells contributes to this phenotype. Therefore, the tight temporal control of spindle elongation and cohesin cleavage assure orchestrated chromosome separation and spindle elongation.

Double-negative feedback between S-phase cyclin-CDK and CKI generates abruptness in the G1/S switch

The G1/S transition is a crucial decision point in the cell cycle. At G1/S, there is an abrupt switch from a state of high cyclin-dependent kinases (CDK) inhibitor (CKI) levels and low S-phase CDK activity to a state of high S-phase CDK activity and degraded CKI. In budding yeast, this transition is triggered by phosphorylation of the Cdk1 inhibitor Sic1 at multiple sites by G1-phase CDK (Cln1,2-Cdk1) and S-phase CDK (Clb5,6-Cdk1) complexes. Using mathematical modeling we demonstrate that the mechanistic basis for the abruptness of the G1/S transition is the highly specific phosphorylation of Sic1 by S-phase CDK complex. This switch is generated by a double-negative feedback loop in which S-CDK1 phosphorylates Sic1, thus targeting it for destruction, and thereby liberating further S-CDK1 from the inhibitory Sic1-S-CDK1 complex. Our model predicts that the abruptness of the switch depends upon a strong binding affinity within the Sic1-S-CDK inhibitory complex. In vitro phosphorylation analysis using purified yeast proteins revealed that free Clb5-Cdk1 can create positive feedback by phosphorylating Sic1 that is bound in the inhibitory complex, and that Sic1 inhibits Clb5-Cdk1 with a sub-nanomolar inhibition constant. Our model also predicts that if the G1-phase CDK complex is too efficient at targeting Sic1 for destruction, then G1/S becomes a smooth and readily reversible transition. We propose that the optimal role for the G1-phase CDK in the switch would not be to act as a kinase activity directly responsible for abrupt degradation of CKI, but rather to act as a priming signal that initiates a positive feedback loop driven by emerging free S-phase CDK.

Enabling Association of the GINS Protein Tetramer with the Mini Chromosome Maintenance (Mcm)2–7 Protein Complex by Phosphorylated Sld2 Protein and Single-stranded Origin DNA

The Cdc45-Mcm2-7-GINS (CMG) complex is the replication fork helicase in eukaryotes. Synthetic lethal with Dpb11-1 (Sld2) is required for the initiation of DNA replication, and the S phase cyclin-dependent kinase (S-CDK) phosphorylates Sld2 in vivo. We purified components of the replication initiation machinery and studied their interactions in vitro. We found that unphosphorylated or CDK-phosphorylated Sld2 binds to the mini chromosome maintenance (Mcm)2-7 complex with similar efficiency. Sld2 interaction with Mcm2-7 blocks the interaction between GINS and Mcm2-7. The interaction between CDK-phosphorylated Sld2 and Mcm2-7 is substantially inhibited by origin single-stranded DNA (ssDNA). Furthermore, origin ssDNA allows GINS to bind to Mcm2-7 in the presence of CDK-phosphorylated Sld2. However, unphosphorylated Sld2 blocks the interaction between GINS and Mcm2-7 even in the presence of origin ssDNA. We identified a mutant of Sld2 that does not bind to DNA. When this mutant is expressed in yeast cells, cell growth is severely inhibited with very slow progression into S phase. We propose a model wherein Sld2 blocks the interaction between GINS and Mcm2-7 in vivo. Once origin ssDNA is extruded from the Mcm2-7 ring and CDK phosphorylates Sld2, the origin ssDNA binds to CDK-phosphorylated Sld2. This event may allow the interaction between GINS and Mcm2-7 in vivo. Thus, CDK phosphorylation of Sld2 may be important to release Sld2 from Mcm2-7, thereby allowing GINS to bind Mcm2-7. Furthermore, origin ssDNA may stimulate the formation of the CMG complex by alleviating inhibitory interactions between Sld2 with Mcm2-7.

The Cdc45·Mcm2–7·GINS Protein Complex in Trypanosomes Regulates DNA Replication and Interacts with Two Orc1-like Proteins in the Origin Recognition Complex

Accurate DNA replication requires a complex interplay of many regulatory proteins at replication origins. The CMG (Cdc45·Mcm2-7·GINS) complex, which is composed of Cdc45, Mcm2-7, and the GINS (Go-Ichi-Ni-San) complex consisting of Sld5 and Psf1 to Psf3, is recruited by Cdc6 and Cdt1 onto origins bound by the heterohexameric origin recognition complex (ORC) and functions as a replicative helicase. Trypanosoma brucei, an early branched microbial eukaryote, appears to express an archaea-like ORC consisting of a single Orc1/Cdc6-like protein. However, unlike archaea, trypanosomes possess components of the eukaryote-like CMG complex, but whether they form an active helicase complex, associate with the ORC, and regulate DNA replication remains unknown. Here, we demonstrated that the CMG complex is formed in vivo in trypanosomes and that Mcm2-7 helicase activity is activated by the association with Cdc45 and the GINS complex in vitro. Mcm2-7 and GINS proteins are confined to the nucleus throughout the cell cycle, whereas Cdc45 is exported out of the nucleus after DNA replication, indicating that nuclear exclusion of Cdc45 constitutes one mechanism for preventing DNA re-replication in trypanosomes. With the exception of Mcm4, Mcm6, and Psf1, knockdown of individual CMG genes inhibits DNA replication and cell proliferation. Finally, we identified a novel Orc1-like protein, Orc1b, as an additional component of the ORC and showed that both Orc1b and Orc1/Cdc6 associate with Mcm2-7 via interactions with Mcm3. All together, we identified the Cdc45·Mcm2-7·GINS complex as the replicative helicase that interacts with two Orc1-like proteins in the unusual origin recognition complex in trypanosomes.

Eukaryotic Origin-Dependent DNA Replication In Vitro Reveals Sequential Action of DDK and S-CDK Kinases

Proper eukaryotic DNA replication requires temporal separation of helicase loading from helicase activation and replisome assembly. Using an invitro assay for eukaryotic origin-dependent replication initiation, we investigated the control of these events. After helicase loading, we found that the Dbf4-dependent Cdc7 kinase (DDK) but not S phase cyclin-dependent kinase (S-CDK) is required for the initial origin recruitment of Sld3 and the Cdc45 helicase-activating protein. Likewise, invivo, DDK drives early-firing-origin recruitment of Cdc45 before activation of S-CDK. After S-CDK activation, a second helicase-activating protein (GINS) and the remainder of the replisome are recruited to the origin. Finally, recruitment of lagging but not leading strand DNA polymerases depends on Mcm10 and DNA unwinding. Our studies identify distinct roles for DDK and S-CDK during helicase activation and support a model in which the leading strand DNA polymerase is recruited prior to origin DNA unwinding and RNA primer synthesis.

Noise Cancellation: Viral Fine Tuning of the Cellular Environment for Its Own Genome Replication

Productive replication of DNA viruses elicits host cell DNA damage responses, which cause both beneficial and detrimental effects on viral replication. In response to the viral productive replication, host cells attempt to attenuate the S-phase cyclin-dependent kinase (CDK) activities to inhibit viral replication. However, accumulating evidence regarding interactions between viral factors and cellular signaling molecules indicate that viruses utilize them and selectively block the downstream signaling pathways that lead to attenuation of the high S-phase CDK activities required for viral replication. In this review, we describe the sophisticated strategy of Epstein-Barr virus to cancel such “noisy” host defense signals in order to hijack the cellular environment.

Sld2 binds to origin single-stranded DNA and stimulates DNA annealing

Sld2 is essential for the initiation of DNA replication, but the mechanism underlying its role in replication is not fully understood. The S-phase cyclin dependent kinase (S-CDK) triggers the association of Sld2 with Dpb11, and a phosphomimetic mutation of Sld2, Sld2T84D, functionally mimics the S-CDK phosphorylated state of Sld2. We report that Sld2T84D binds directly to the single-stranded (ss) DNA of two different origins of replication, and S-CDK phosphorylation of Sld2 stimulates the binding of Sld2 to origin ssDNA. Sld2T84D binds to a thymine-rich ssDNA region of the origin ARS1, and substitution of ARS1 thymines with adenines completely disrupts binding of Sld2T84D. Sld2T84D enhances the ability of origin ssDNA to pulldown Dpb11, and Sld2 binding to origin ssDNA may be important to allow Sld2 and Dpb11 to associate with origin DNA. We also report that Sld2T84D anneals ssDNA of an origin sequence. Dpb11 anneals ssDNA to low levels, and the addition of Sld2T84D with Dpb11 results in higher annealing activity than that of either protein alone. Sld2-stimulated annealing may be important for maintaining genome stability during the initiation of DNA replication.

Cdk2 Is Required for p53-Independent G2/M Checkpoint Control

The activation of phase-specific cyclin-dependent kinases (Cdks) is associated with ordered cell cycle transitions. Among the mammalian Cdks, only Cdk1 is essential for somatic cell proliferation. Cdk1 can apparently substitute for Cdk2, Cdk4, and Cdk6, which are individually dispensable in mice. It is unclear if all functions of non-essential Cdks are fully redundant with Cdk1. Using a genetic approach, we show that Cdk2, the S-phase Cdk, uniquely controls the G2/M checkpoint that prevents cells with damaged DNA from initiating mitosis. CDK2-nullizygous human cells exposed to ionizing radiation failed to exclude Cdk1 from the nucleus and exhibited a marked defect in G2/M arrest that was unmasked by the disruption of P53. The DNA replication licensing protein Cdc6, which is normally stabilized by Cdk2, was physically associated with the checkpoint regulator ATR and was required for efficient ATR-Chk1-Cdc25A signaling. These findings demonstrate that Cdk2 maintains a balance of S-phase regulatory proteins and thereby coordinates subsequent p53-independent G2/M checkpoint activation.

Temporal control of the dephosphorylation of Cdk substrates by mitotic exit pathways in budding yeast

The temporal phosphorylation of cell cycle-related proteins by cyclin-dependent kinases (Cdks) is critical for the correct order of cell cycle events. In budding yeast, CDC28 encodes the only Cdk and its association with various cyclins governs the temporal phosphorylation of Cdk substrates. S-phase Cdk substrates are phosphorylated earlier than mitotic Cdk substrates, which ensures the sequential order of DNA synthesis and mitosis. However, it remains unclear whether Cdk substrates are dephosphorylated in temporally distinct windows. Cdc14 is a conserved protein phosphatase responsible for the dephosphorylation of Cdk substrates. In budding yeast, FEAR (Cdc14 early anaphase release) and MEN (mitotic exit network) activate phosphatase Cdc14 by promoting its release from the nucleolus in early and late anaphase, respectively. Here, we show that the sequential Cdc14 release and the distinct degradation timing of different cyclins provides the molecular basis for the differential dephosphorylation windows of S-phase and mitotic cyclin substrates. Our data also indicate that FEAR-induced dephosphorylation of S-phase Cdk substrates facilitates anaphase progression, revealing an extra layer of mitotic regulation.

Akt-mediated phosphorylation of CDK2 regulates its dual role in cell cycle progression and apoptosis

Here, we show that CDK2, an S-phase cyclin-dependent kinase, is a novel target for Akt during cell cycle progression and apoptosis. Akt phosphorylates CDK2 at threonine 39 residue both in vitro and in vivo. Although CDK2 threonine 39 phosphorylation mediated by Akt enhances cyclin-A binding, it is dispensable for its basal binding and the kinase activity. In addition, for the first time, we report a transient nucleo-cytoplasmic shuttling of Akt during specific stages of the cell cycle, in particular during the late S and G2 phases. The Akt that is re-localized to the nucleus phosphorylates CDK2 and causes the temporary cytoplasmic localization of the CDK2-cyclin-A complex. The CDK2 cytoplasmic redistribution is required for cell progression from S to G2-M phase, because the CDK2 T39A mutant, which lacks the phosphorylation site and is defective in cytoplasmic localization, severely affects cell cycle progression at the transition from S to G2-M. Interestingly, we also show that the Akt/CDK2 pathway is constitutively activated by some anticancer drugs, such as methotrexate and docetaxel, and under these conditions it promotes, rather than represses, cell death. Thus, the constitutive activation of the Akt/CDK2 pathway and changed subcellular localization promotes apoptosis. By contrast, the transient, physiological Akt/CDK2 activation is necessary for cell cycle progression.

Cdc28–Clb5 (CDK-S) and Cdc7–Dbf4 (DDK) collaborate to initiate meiotic recombination in yeast

S-phase cyclin-dependent kinase Cdc28-Clb5 (CDK-S) and Dbf4-dependent kinase Cdc7-Dbf4 (DDK) are highly conserved kinases well known for their roles in the initiation of DNA replication. CDK-S is also essential for initiation of meiotic recombination because it phosphorylates Ser30 of Mer2, a meiosis-specific double-strand break (DSB) protein. This work shows that the phosphorylation of Mer2 Ser30 by CDK-S primes Mer2 for subsequent phosphorylation by DDK on Ser29, creating a negatively charged "patch" necessary for DSB formation. CDK-S and DDK phosphorylation of Mer2 S30 and S29 can be bypassed by phosphomimetic amino acids, but break formation under these conditions is still dependent on DDK and CDK-S activity. Coordination between premeiotic S and DSB formation may be achieved by using CDK-S and DDK to initiate both processes. Many other proteins important for replication, recombination, repair, and chromosome segregation contain combination DDK/CDK sites, raising the possibility that this is a common regulatory mechanism.

Phosphorylation of Sld2 and Sld3 by cyclin-dependent kinases promotes DNA replication in budding yeast

Cyclin-dependent kinases (CDKs) drive major cell cycle events including the initiation of chromosomal DNA replication. We identified two S phase CDK (S-CDK) phosphorylation sites in the budding yeast Sld3 protein that, together, are essential for DNA replication. Here we show that, when phosphorylated, these sites bind to the amino-terminal BRCT repeats of Dpb11. An Sld3-Dpb11 fusion construct bypasses the requirement for both Sld3 phosphorylation and the N-terminal BRCT repeats of Dpb11. Co-expression of this fusion with a phospho-mimicking mutant in a second essential CDK substrate, Sld2, promotes DNA replication in the absence of S-CDK. Therefore, Sld2 and Sld3 are the minimal set of S-CDK targets required for DNA replication. DNA replication in cells lacking G1 phase CDK (G1-CDK) required expression of the Cdc7 kinase regulatory subunit, Dbf4, as well as Sld2 and Sld3 bypass. Our results help to explain how G1- and S-CDKs promote DNA replication in yeast.