Cdc2 Kinase Research Articles

Transcription factor Atf1-dependent degradation of the mitotic cyclin Cdc13 is regulated by multiple factors in Schizosaccharomyces pombe.

The bZIP transcription factor Atf1 is a key player in the transcriptional programme of Schizosaccharomyces pombe cell cycle. It also controls both expression and degradation of mitotic cyclin Cdc13. Temporal regulation of these opposing functions of Atf1 is critical for fidelity of cell division. Our investigations revealed that an increase in the activity of mitogen-activated protein kinase (MAPK) Spc1 during mitotic exit and the consequent phosphorylation of Atf1 along with the prevailing high activity of cyclin-dependent kinase Cdc2 regulate Cdc13 degradation. Our results also indicate the possibility of a complex interplay between Cdc2 inhibitory kinase Wee1, the anaphase-promoting complex and Atf1 during mitotic exit. These observations provide evidence of new regulatory mechanisms of mitotic exit.

WEE1 inhibition augments CDC7 (DDK) inhibitor-induced cell death in Ewing sarcoma by forcing premature mitotic entry and mitotic catastrophe.

Ewing sarcoma is an aggressive childhood cancer for which treatment options remain limited and toxic. There is an urgent need for the identification of novel therapeutic strategies. Our group has recently shown that Ewing cells rely on the S-phase kinase CDC7 (DDK) to maintain replication rates and cell viability and that DDK inhibition causes an increase in the phosphorylation of CDK1 and a significant delay in mitotic entry. Here, we expand on our previous findings and show that DDK inhibitor-induced mitotic entry delay is dependent upon WEE1 kinase. Specifically, WEE1 phosphorylates CDK1 and prevents mitotic entry upon DDK inhibition due to the presence of under-replicated DNA, potentially limiting the cytotoxic effects of DDK inhibition. To overcome this, we combined the inhibition of DDK with the inhibition of WEE1 and found that this results in elevated levels of premature mitotic entry, mitotic catastrophe, and apoptosis. Importantly, we have found that DDK and WEE1 inhibitors display a synergistic relationship with regards to reducing cell viability of Ewing sarcoma cells. Interestingly, the cytotoxic nature of this combination can be suppressed by the inhibition of CDK1 or microtubule polymerization, indicating that mitotic progression is required to elicit the cytotoxic effects. This is the first study to display the potential of utilizing the combined inhibition of DDK and WEE1 for the treatment of cancer. We believe this will offer a potential therapeutic strategy for the treatment of Ewing sarcoma as well as other tumor types that display sensitivity to DDK inhibitors.

A function for ataxia telangiectasia and Rad3-related (ATR) kinase in cytokinetic abscission

SummaryAbscission, the final stage of cytokinesis, occurs when the cytoplasmic canal connecting two emerging daughter cells is severed either side of a large proteinaceous structure, the midbody. Here, we expand the functions of ATR to include a cell-cycle-specific role in abscission, which is required for genome stability. All previously characterized roles for ATR depend upon its recruitment to replication protein A (RPA)-coated single-stranded DNA (ssDNA). However, we establish that in each cell cycle ATR, as well as ATRIP, localize to the midbody specifically during late cytokinesis and independently of RPA or detectable ssDNA. Rather, midbody localization and ATR-dependent regulation of abscission requires the known abscission regulator-charged multivesicular body protein 4C (CHMP4C). Intriguingly, this regulation is also dependent upon the CDC7 kinase and the known ATR activator ETAA1. We propose that in addition to its known RPA-ssDNA-dependent functions, ATR has further functions in preventing premature abscission.

The structural basis of Cdc7-Dbf4 kinase dependent targeting and phosphorylation of the MCM2-7 double hexamer

The controlled assembly of replication forks is critical for genome stability. The Dbf4-dependent Cdc7 kinase (DDK) initiates replisome assembly by phosphorylating the MCM2-7 replicative helicase at the N-terminal tails of Mcm2, Mcm4 and Mcm6. At present, it remains poorly understood how DDK docks onto the helicase and how the kinase targets distal Mcm subunits for phosphorylation. Using cryo-electron microscopy and biochemical analysis we discovered that an interaction between the HBRCT domain of Dbf4 with Mcm2 serves as an anchoring point, which supports binding of DDK across the MCM2-7 double-hexamer interface and phosphorylation of Mcm4 on the opposite hexamer. Moreover, a rotation of DDK along its anchoring point allows phosphorylation of Mcm2 and Mcm6. In summary, our work provides fundamental insights into DDK structure, control and selective activation of the MCM2-7 helicase during DNA replication. Importantly, these insights can be exploited for development of novel DDK inhibitors.

Regulation of CLB6 expression by the cytoplasmic deadenylase Ccr4 through its coding and 3’ UTR regions

RNA stability control contributes to the proper expression of gene products. Messenger RNAs (mRNAs) in eukaryotic cells possess a 5’ cap structure and the 3’ poly(A) tail which are important for mRNA stability and efficient translation. The Ccr4-Not complex is a major cytoplasmic deadenylase and functions in mRNA degradation. The CLB1-6 genes in Saccharomyces cerevisiae encode B-type cyclins which are involved in the cell cycle progression together with the cyclin-dependent kinase Cdc28. The CLB genes consist of CLB1/2, CLB3/4, and CLB5/6 whose gene products accumulate at the G2-M, S-G2, and late G1 phase, respectively. These Clb protein levels are thought to be mainly regulated by the transcriptional control and the protein stability control. Here we investigated regulation of CLB1-6 expression by Ccr4. Our results show that all CLB1-6 mRNA levels were significantly increased in the ccr4Δ mutant compared to those in wild-type cells. Clb1, Clb4, and Clb6 protein levels were slightly increased in the ccr4Δ mutant, but the Clb2, Clb3, and Clb5 protein levels were similar to those in wild-type cells. Since both CLB6 mRNA and Clb6 protein levels were most significantly increased in the ccr4Δ mutant, we further analyzed the cis-elements for the Ccr4-mediated regulation within CLB6 mRNA. We found that there were destabilizing sequences in both coding sequence and 3’ untranslated region (3’ UTR). The destabilizing sequences in the coding region were found to be both within and outside the sequences corresponding the cyclin domain. The CLB6 3’ UTR was sufficient for mRNA destabilization and decrease of the reporter GFP gene and this destabilization involved Ccr4. Our results suggest that CLB6 expression is regulated by Ccr4 through the coding sequence and 3’ UTR of CLB6 mRNA.

CDC7-independent G1/S transition revealed by targeted protein degradation.

The entry of mammalian cells into the DNA synthesis phase (S phase) represents a key event in cell division1. According to current models of the cell cycle, the kinase CDC7 constitutes an essential and rate-limiting trigger of DNA replication, acting together with the cyclin-dependent kinase CDK2. Here we show that CDC7 is dispensable for cell division of many different cell types, as determined using chemical genetic systems that enableacute shutdown of CDC7 in cultured cells and in live mice. We demonstrate that another cell cycle kinase, CDK1, is also active during G1/S transition both in cycling cells and in cells exiting quiescence. We show thatCDC7 and CDK1 perform functionally redundant roles during G1/S transition, and at least one of these kinases must be present to allow S-phase entry. These observations revise our understanding of cell cycle progression by demonstrating that CDK1 physiologically regulates two distinct transitions during cell division cycle, whereas CDC7 hasa redundant functionin DNA replication.

Structural Insight into the MCM double hexamer activation by Dbf4-Cdc7 kinase

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.

CDC7 kinase (DDK) inhibition disrupts DNA replication leading to mitotic catastrophe in Ewing sarcoma

Ewing sarcoma is the second most common bone malignancy in children and adolescents. In recent years, a large body of evidence has emerged that suggests Ewing tumors harbor large amounts of replication stress (RS). CDC7, also known as DDK (DBF4-dependent kinase), is a serine/threonine kinase that is involved in a diverse array of cellular functions including the regulation of DNA replication initiation and activation of the RS response. Due to DDK’s diverse roles during replication, coupled with the fact that there is an increased level of RS within Ewing tumors, we hypothesized that Ewing sarcoma cells would be particularly vulnerable to DDK inhibition. Here, we report that DDK inhibition resulted a significant reduction in cell viability and the induction of apoptosis, specifically in Ewing sarcoma cells. Treatment with DDK inhibitors dramatically reduced the rate of replication, prolonged S-phase, and led to a pronounced increase in phospho-CDC2 (Y15), indicating delay of mitotic entry. The induction of cell death corresponded to mitotic exit and G1 entry, suggesting improper mitotic progression. In accordance with this, we find that DDK inhibition caused premature mitotic entry resulting in mitotic abnormalities such as anaphase bridges, lagging chromosomes, and cells with >2 poles in Ewing sarcoma cells. This abnormal progression through mitosis resulted in mitotic catastrophe as evidenced by the formation of micronuclei and induction of DNA damage. Together, these findings suggest that DDK activity is required for the faithful and timely completion of DNA replication in Ewing cells and that DDK inhibition may present a viable therapeutic strategy for the treatment of Ewing sarcoma.

Chiffon triggers global histone H3 acetylation and expression of developmental genes in Drosophila embryos.

ABSTRACTThe histone acetyltransferase Gcn5 is critical for gene expression and development. In Drosophila, Gcn5 is part of four complexes (SAGA, ATAC, CHAT and ADA) that are essential for fly viability and have key roles in regulating gene expression. Here, we show that although the SAGA, ADA and CHAT complexes play redundant roles in embryonic gene expression, the insect-specific CHAT complex uniquely regulates expression of a subset of developmental genes. We also identify a substantial decrease in histone acetylation in chiffon mutant embryos that exceeds that observed in Ada2b, suggesting broader roles for Chiffon in regulating histone acetylation outside of the Gcn5 complexes. The chiffon gene encodes two independent polypeptides that nucleate formation of either the CHAT or Dbf4-dependent kinase (DDK) complexes. DDK includes the cell cycle kinase Cdc7, which is necessary for maternally driven DNA replication in the embryo. We identify a temporal switch between the expression of these chiffon gene products during a short window during the early nuclear cycles in embryos that correlates with the onset of zygotic genome activation, suggesting a potential role for CHAT in this process. This article has an associated First Person interview with the first author of the paper.

The Spo13/Meikin pathway confines the onset of gamete differentiation to meiosis II in yeast

Sexual reproduction requires genome haploidization by the two divisions of meiosis and a differentiation program to generate gametes. Here, we have investigated how sporulation, the yeast equivalent of gamete differentiation, is coordinated with progression through meiosis. Spore differentiation is initiated at metaphase II when a membrane‐nucleating structure, called the meiotic plaque, is assembled at the centrosome. While all components of this structure accumulate already at entry into meiosis I, they cannot assemble because centrosomes are occupied by Spc72, the receptor of the γ‐tubulin complex. Spc72 is removed from centrosomes by a pathway that depends on the polo‐like kinase Cdc5 and the meiosis‐specific kinase Ime2, which is unleashed by the degradation of Spo13/Meikin upon activation of the anaphase‐promoting complex at anaphase I. Meiotic plaques are finally assembled upon reactivation of Cdk1 at entry into metaphase II. This unblocking‐activation mechanism ensures that only single‐copy genomes are packaged into spores and might serve as a paradigm for the regulation of other meiosis II‐specific processes.

Structural mechanism for the selective phosphorylation of DNA-loaded MCM double hexamers by the Dbf4-dependent kinase

Loading of the eukaryotic replicative helicase onto replication origins involves two MCM hexamers forming a double hexamer (DH) around duplex DNA. During S phase, helicase activation requires MCM phosphorylation by Dbf4-dependent kinase (DDK), comprising Cdc7 and Dbf4. DDK selectively phosphorylates loaded DHs, but how such fidelity is achieved is unknown. Here, we determine the cryogenic electron microscopy structure of Saccharomyces cerevisiae DDK in the act of phosphorylating a DH. DDK docks onto one MCM ring and phosphorylates the opposed ring. Truncation of the Dbf4 docking domain abrogates DH phosphorylation, yet Cdc7 kinase activity is unaffected. Late origin firing is blocked in response to DNA damage via Dbf4 phosphorylation by the Rad53 checkpoint kinase. DDK phosphorylation by Rad53 impairs DH phosphorylation by blockage of DDK binding to DHs, and also interferes with the Cdc7 active site. Our results explain the structural basis and regulation of the selective phosphorylation of DNA-loaded MCM DHs, which supports bidirectional replication.

Evidences for Red Pigment Concentrating Hormone (RPCH) and Beta-Pigment Dispersing Hormone (β-PDH) Inducing Oocyte Meiotic Maturation in the Chinese Mitten Crab, Eriocheir sinensis.

Red pigment concentrating hormone (RPCH) and pigment dispersing hormone (PDH) are crustacean neuropeptides involved in broad physiological processes including body color changes, circadian rhythm, and ovarian growth. In this study, the full-length cDNA of RPCH and PDH were identified from the brain of the Chinese mitten crab Eriocheir sinensis. The deduced RPCH and PDH mature peptides shared identical sequence to the adipokinetic hormone/RPCH peptides family and the β-PDH isoforms and were designated as Es-RPCH and Es-β-PDH, respectively. Es-RPCH and Es-β-PDH transcripts were distributed in the brain and eyestalks. The positive signals of Es-RPCH and Es-β-PDH were localized in the neuronal clusters 6, 8, 9, 10, and 17 of the brain as revealed by in situ hybridization. The expression level of Es-RPCH and Es-β-PDH mRNA in nervous tissues were all significantly increased at vitellogenic stage, and then decreased at the final meiotic maturation stage. The administrated with synthesized Es-RPCH peptide results in germinal vesicles shift toward the plasma membrane in vitellogenic oocyte, and significant decrease of the gonad-somatic index (GSI) and mean oocyte diameter as well as the expression of vitellogenin mRNA at 30 days post injection in vivo. Similar results were also found when injection of the Es-β-PDH peptide. In vitro culture demonstrated that Es-RPCH and Es-β-PDH induced germinal vesicle breakdown of the late vitellogenic oocytes. Comparative ovarian transcriptome analysis indicated that some reproduction/meiosis-related genes such as cdc2 kinase, cyclin B, 5-HT-R and retinoid-X receptor were significantly upregulated in response to Es-RPCH and Es-β-PDH treatments. Taken together, these results provided the evidence for the inductive effect of Es-RPCH and Es-β-PDH on the oocyte meiotic maturation in E. sinensis.

Discovery of AS-0141, a Potent and Selective Inhibitor of CDC7 Kinase for the Treatment of Solid Cancers.

CDC7, a serine-threonine kinase, plays conserved and important roles in regulation of DNA replication and has been recognized as a potential anticancer target. We report here the optimization of a series of furanone analogues starting from compound 1 with a focus on ADME properties suitable for clinical development. By replacing the 2-chlorobenzene moiety in 1 with various aliphatic groups, we identified compound 24 as a potent CDC7 inhibitor with excellent kinase selectivity and favorable oral bioavailability in multiple species. Oral administration of 24 demonstrated robust in vivo antitumor efficacy in a colorectal cancer xenograft model. Compound 24 (AS-0141) is currently in phase I clinical trials for the treatment of solid cancers.

Phosphorylation of luminal region of the SUN-domain protein Mps3 promotes nuclear envelope localization during meiosis.

During meiosis, protein ensembles in the nuclear envelope (NE) containing SUN- and KASH-domain proteins, called linker nucleocytoskeleton and cytoskeleton (LINC) complex, promote the chromosome motion. Yeast SUN-domain protein, Mps3, forms multiple meiosis-specific ensembles on NE, which show dynamic localisation for chromosome motion; however, the mechanism by which these Mps3 ensembles are formed during meiosis remains largely unknown. Here, we showed that the cyclin-dependent protein kinase (CDK) and Dbf4-dependent Cdc7 protein kinase (DDK) regulate meiosis-specific dynamics of Mps3 on NE, particularly by mediating the resolution of Mps3 clusters and telomere clustering. We also found that the luminal region of Mps3 juxtaposed to the inner nuclear membrane is required for meiosis-specific localisation of Mps3 on NE. Negative charges introduced by meiosis-specific phosphorylation in the luminal region of Mps3 alter its interaction with negatively charged lipids by electric repulsion in reconstituted liposomes. Phospho-mimetic substitution in the luminal region suppresses the localisation of Mps3 via the inactivation of CDK or DDK. Our study revealed multi-layered phosphorylation-dependent regulation of the localisation of Mps3 on NE for meiotic chromosome motion and NE remodelling.

Abstract 2051: Inhibiting CDC7 kinase for the treatment of Ewing sarcoma

Abstract Ewing Sarcoma is a malignant tumor of the bone and soft tissue that is primarily driven by the fusion protein EWS-FLI1. With no current targeted therapy, treatment options remain limited and toxic. Recent reports have shown that Ewing tumors harbor large amounts of transcription-associated replication stress and that this offers an attractive therapeutic intervention point. Our group has shown that inhibition of the kinase complex DDK (CDC7-DBF4) results in apoptosis and a significant reduction in cell viability in EWS-FLI1-harboring Ewing's sarcoma cell lines. Also, low dose DDK inhibition in Ewing's sarcoma cells caused an S/G2 cell cycle accumulation at 24 hours, followed by re-entry into G1 and appearance of sub-G1 DNA. This suggests a failure to properly progress through S-phase followed by premature mitotic entry and mitotic catastrophe resulting in cell death. To this end, there was a significant increase in the formation of micronuclei and other aberrant mitotic structures upon DDK inhibition in Ewing's cells. Interestingly, there was minimal signs of mitotic accumulation, despite clear evidence of mitotic stress, indicating a failure to properly enforce a G2-M checkpoint. Importantly, these phenotypes were not mimicked with the use of the CDK2 inhibitor Roscotivine, suggesting an origin firing-independent basis for the sensitivity to DDK inhibition. Furthermore, we show that DDK inhibition synergizes with the TOP1 inhibitor Camptothecin and with the PARP1 inhibitor Talazoparib to kill Ewing Sarcoma cells in vitro. We have hypothesized this to be due to DDK's potential role in replication fork instability/restart. Together, these results suggest that Ewing sarcoma cells rely on the activity of DDK to maintain cell viability independently from its role in the regulation of replication origin initiation and suggest that DDK inhibition may prove to be a viable treatment option for patients with Ewing's sarcoma. Citation Format: Jeffrey C. Martin. Inhibiting CDC7 kinase for the treatment of Ewing sarcoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2051.

Abstract 1277: Discovery of novel CDC7 inhibitors that disrupt cell cycle dynamics and show anti-proliferative effects in cancer cells

Abstract Introduction: CDC7 is a serine/threonine protein kinase that phosphorylates the MCM2-7 helicase complex, a required step in DNA replication initiation. CDC7 has emerged as an attractive target for cancer treatment because of high expression in a number of tumors (e.g. ovarian, lung, and oral) which is thought to be linked to their proliferative capacity and ability to bypass DNA damage responses. Consistent with this, disruption of CDC7 activity in cancer cells results in delayed DNA replication, mitotic abnormalities and cell death whereas non-transformed, p53 wildtype cells are protected from cytotoxicity due to G1 cell cycle arrest. Due to the low ATP Km of CDC7, very potent inhibitor molecules are required to effectively block CDC7 activity and drive cancer cells into apoptosis. We have identified novel potent and selective CDC7 inhibitors targeting the ATP binding site that are active in biophysical, biochemical and cellular assays as well as in vivo CDX models. Results: Our lead compounds show potent picomolar (pM) inhibition of CDC7 in a biochemical kinase activity assay, pM affinity in SPR assay and complete inhibition of MCM2 (S53) phosphorylation in COLO205, A427, MV-4-11 and SW48 cancer cell lines. In a broad kinase selectivity panel, the novel inhibitors showed good selectivity for CDC7 kinase. Mechanistic studies show that our CDC7 inhibitors induced apoptosis, disrupted DNA replication and cell cycle dynamics with accumulation of polyploid cells after 48 h of treatment of cancer cells with minimal effects on human fibroblast cell lines. Our compounds have shown potent anti-proliferative and cytotoxic effects in a panel of more than a 100 cancer cell lines of varying origin including COLO205, SW48, A427, MOLM-13, and SUM149. Comparison of CDC7 inhibitors with other oncology drugs in a panel of cancer cell lines revealed a unique mechanism of action. In vivo, our compounds reduced tumor cell MCM2 (S53) phosphorylation in the mouse COLO205 xenograft model and showed strong tumor growth inhibition. We have also examined the effect of CDC7 inhibitors on cancer cell proliferation in combination with other anti-cancer agents, including other DNA damage response (DDR) targeting agents. Conclusions: We have identified novel potent ATP-competitive CDC7 inhibitors that show target engagement in cells and CDX tumors and have shown strong inhibition of cancer cell proliferation in vitro and tumor growth in vivo. CDC7 inhibitors show promise for use in combination with other targeted therapies for the treatment of cancers of varying origin. Citation Format: Lyuben Tsvetkov, Adam Levinson, Xianhai Huang, Sayan Mondal, Jeff Bell, Lin Tang, Robert Pelletier, Karen Dingley, Nick Boyles, Jackson Chief Elk, Leah Frye, Alan Futran, Phani Ghanakota, Jeremy Greenwood, George Lai, Sarah Silvergleid, Wu Yin, Hamish Wright, Karen Akinsanya, Wayne Tang, Kristian Jensen. Discovery of novel CDC7 inhibitors that disrupt cell cycle dynamics and show anti-proliferative effects in cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1277.

A TOR (target of rapamycin) and nutritional phosphoproteome of fission yeast reveals novel targets in networks conserved in humans.

Fluctuations in TOR, AMPK and MAP-kinase signalling maintain cellular homeostasis and coordinate growth and division with environmental context. We have applied quantitative, SILAC mass spectrometry to map TOR and nutrient-controlled signalling in the fission yeast Schizosaccharomyces pombe. Phosphorylation levels at more than 1000 sites were altered following nitrogen stress or Torin1 inhibition of the TORC1 and TORC2 networks that comprise TOR signalling. One hundred and thirty of these sites were regulated by both perturbations, and the majority of these (119) new targets have not previously been linked to either nutritional or TOR control in either yeasts or humans. Elimination of AMPK inhibition of TORC1, by removal of AMPKα (ssp2::ura4+), identified phosphosites where nitrogen stress-induced changes were independent of TOR control. Using a yeast strain with an ATP analogue-sensitized Cdc2 kinase, we excluded sites that were changed as an indirect consequence of mitotic control modulation by nitrogen stress or TOR signalling. Nutritional control of gene expression was reflected in multiple targets in RNA metabolism, while significant modulation of actin cytoskeletal components points to adaptations in morphogenesis and cell integrity networks. Reduced phosphorylation of the MAPKK Byr1, at a site whose human equivalent controls docking between MEK and ERK, prevented sexual differentiation when resources were sparse but not eliminated.

The High Mobility Group I/Y Protein Is Hypophosphorylated in Endoreduplicating Maize Endosperm Cells and Is Involved in Alleviating Histone H1-mediated Transcriptional Repression

During maize endosperm development, cells shift from a mitotic cycle to endoreduplication, driving the massive synthesis of storage proteins (zeins) and starch. In this developmental context, we studied changes in expression levels of histone H1 and high mobility group I/Y (HMG-I/Y), two chromatin architectural proteins that are known to affect gene transcription. Almost no change was found in the level of histone H1 during endosperm development, despite a dramatic increase in DNA content (endoreduplication); hence, the histone H1/DNA ratio decreased substantially. Concurrently with a reduction in the Cdc2 kinase activity at the shift to endoreduplication, significant changes were found in the level and mobility of the HMG-I/Y protein; the faster migrating forms were, at least partly, hypophosphorylated. Purified maize HMG-I/Y protein was found to be phophorylated in vitro by the Cdc2 kinase and bound efficiently to the gamma-zein promoter AT-rich tract (gammaZ-AT). Using an in vitro transcription assay, we demonstrated the capability of the maize HMG-I/Y protein to relieve the inhibitory effect exerted by histone H1 on templates containing the gammaZ-AT sequence. These data suggest that during maize endosperm development transcription and perhaps replication are controlled, at least partly, by the activity of the Cdc2 kinase and the interplay between histone H1 and HMG-I/Y proteins.

Development of imidazolone based angiotensin II receptor type I inhibitor small molecule as a chemotherapeutic agent for cell cycle inhibition

Cell cycle inhibitors are considered as hallmark strategy for cancer treatment due to their relatively higher selectivity and efficacy on various cancer types in comparison to cytotoxic agents. Small molecules target dividing cells in G1/S, G2 or M phases of cells to arrest and eventually trigger cancer cells to enter apoptosis and/or inhibit tumor growth. Cell cycle arrest at G2/M phase is a widely used approach to inhibit proliferating cancer cells. We report a novel angiotensin II receptor type I (AT1R) antagonist that also targets CDC2 (CDK1) kinase thereby showing a putative anti-cancer activity. The molecule named 19D was tested in various cancer cell lines at different concentrations. Molecular mechanisms of action triggered by the treatment of 19D were investigated for anti-cancer activity at cellular and molecular level. 19D molecule showed a potent cell cycle arrest with anti-proliferation activity. Cells treated with 19D showed nuclear deteriorations and apoptotic induction on cancer cells. Moreover, the mechanism of cell cycle inhibition was revealed as G2/M arrest via suppressing the CDC2 kinase cell cycle check point inhibitor. In conclusion, 19D (an AT1R antagonist) is a novel small molecule/imidazolone derivative which has been demonstrated as a fairly potent anti-cancer molecule. Abbreviations: AT1R: angiotensin II receptor type 1; BSA: bovine serum albumine; CDC2: cyclin-dependent kinase 1; CDK: cyclin-dependent kinase; DMEM: Dulbecco’s modified eagle medium; DMSO: dimethylsulfoxide; EDTA: ethylenediaminetetraacetic acid; FBS: fetal bovine serum; HBSS: Hank’s balanced salt solution; IC50: half maximal inhibitory concentration; PBS-T: phosphate buffered saline – TritonX; PBS: phosphate buffered saline; PDB: protein data bank; SDS: sodium dodecyl sulfate; TBS-T: Tris buffered saline – TritonX

Post-translational Regulation of DNA Polymerase η, a Connection to Damage-Induced Cohesion in Saccharomyces cerevisiae.

Double-strand breaks that are induced postreplication trigger establishment of damage-induced cohesion in Saccharomyces cerevisiae, locally at the break site and genome-wide on undamaged chromosomes. The translesion synthesis polymerase, polymerase η, is required for generation of damage-induced cohesion genome-wide. However, its precise role and regulation in this process is unclear. Here, we investigated the possibility that the cyclin-dependent kinase Cdc28 and the acetyltransferase Eco1 modulate polymerase η activity. Through in vitro phosphorylation and structure modeling, we showed that polymerase η is an attractive substrate for Cdc28. Mutation of the putative Cdc28-phosphorylation site Ser14 to Ala not only affected polymerase η protein level, but also prevented generation of damage-induced cohesion in vivo. We also demonstrated that Eco1 acetylated polymerase η in vitro. Certain nonacetylatable polymerase η mutants showed reduced protein level, deficient nuclear accumulation, and increased ultraviolet irradiation sensitivity. In addition, we found that both Eco1 and subunits of the cohesin network are required for cell survival after ultraviolet irradiation. Our findings support functionally important Cdc28-mediated phosphorylation, as well as post-translational modifications of multiple lysine residues that modulate polymerase η activity, and provide new insights into understanding the regulation of polymerase η for damage-induced cohesion.