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

AbstractLoading 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.

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WEE1 kinase inhibitor AZD1775 induces CDK1 kinase-dependent origin firing in unperturbed G1- and S-phase cells

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1915108116 ◽

2019 ◽

Vol 116 (48) ◽

pp. 23891-23893 ◽

Cited By ~ 8

Author(s):

Tatiana N. Moiseeva ◽

Chenao Qian ◽

Norie Sugitani ◽

Hatice U. Osmanbeyoglu ◽

Christopher J. Bakkenist

Keyword(s):

Cell Cycle ◽

Clinical Trials ◽

Cell Cycle Regulation ◽

Kinase Inhibitor ◽

S Phase ◽

Origin Firing ◽

Replicative Helicase ◽

Origin Licensing ◽

Wee1 Kinase ◽

Cycle Regulation

WEE1 kinase is a key regulator of the G2/M transition. The WEE1 kinase inhibitor AZD1775 (WEE1i) induces origin firing in replicating cells. We show that WEE1i induces CDK1-dependent RIF1 phosphorylation and CDK2- and CDC7-dependent activation of the replicative helicase. WEE1 suppresses CDK1 and CDK2 kinase activities to regulate the G1/S transition after the origin licensing is complete. We identify a role for WEE1 in cell cycle regulation and important effects of AZD1775, which is in clinical trials.

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Cdc7 inhibition as a novel approach for pancreas cancer therapy.

Journal of Clinical Oncology ◽

10.1200/jco.2013.31.15_suppl.e15059 ◽

2013 ◽

Vol 31 (15_suppl) ◽

pp. e15059-e15059

Author(s):

Mark G. Frattini ◽

Lucia Regales ◽

Ruth Santos ◽

Diana Carrillo

Keyword(s):

Cell Cycle ◽

Pancreatic Cancer ◽

Dna Damage ◽

Cell Viability ◽

Cell Cycle Arrest ◽

Western Blotting ◽

Kinase Activity ◽

S Phase ◽

Cycle Arrest ◽

Cdc7 Kinase

e15059 Background: Pancreatic cancer is the fourth leading cause of cancer death in the USA. In 2012, 43,920 people will be diagnosed and 37,390 people will die of this disease. 95% of tumors reveal loss of the p16 protein, a regulator of the G1 to S phase transition. Cdc7 is a conserved kinase required for the initiation of DNA replication, is a target of the S-phase checkpoint, and has a role in controlling the DNA damage response. Downregulation of Cdc7 kinase activity resulted in slowing of S-phase and cell cycle arrest followed by accumulation of DNA damage. Cdc7 has been shown to be over-expressed in many different tumors including the majority of solid and liquid tumors. In our laboratory a novel natural product small molecule inhibitor (MSK-777) has been identified, developed and shown to be efficacious in cell based cytotoxicity assays and multiple animal models of cancer. Methods: We have examined the efficacy of Cdc7 kinase inhibition as a therapeutic approach for pancreatic cancer by examining the sensitivity of MSK-777 in Capan-1, BxPC3, and PANC-1 cell lines. These cells were treated with MSK-777, control (DMSO), or hydroxyurea and collected for viable cell counts, fluorescence-activated cell sorting (FACS), and western blotting. Results: Cell viability analyses revealed that MSK-777 had a dramatic effect after 24 hours, reducing cell viability to less then 20% in BxPC3 cells. FACS results demonstrated that MSK-777 exposure resulted in cell cycle arrest at G1/S in Capan-1 and PANC-1 cells by 48 hours while BxPC3 cells showed a significant sub-G1 population by 24 hours, indicating apoptotic cell death. Western blotting showed that in BxPC3 cells phosphorylation of the mini-chromosome maintenance 2 protein (Mcm2) disappeared by 24 hours, indicating inactivation of the helicase that unwinds the strands of DNA during replication. Western blots of Capan-1 and PANC-1 cells showed lower levels of phosphorylated Mcm2 by 48 hours. Conclusions: We are currently examining the efficacy of MSK-777 in mouse models of orthotopically injected pancreatic cancer cells. Based on these collective results, inhibition of Cdc7 kinase activity with MSK-777 represents a novel and promising therapy for this deadly disease.

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Structural basis for repurposing a 100-years-old drug suramin for treating COVID-19

10.21203/rs.3.rs-99513/v1 ◽

2020 ◽

Author(s):

H. Eric Xu ◽

Wanchao Yin ◽

Xiaodong Luan ◽

Zhihai Li ◽

Leike Zhang ◽

...

Keyword(s):

Active Site ◽

Binding Sites ◽

Potent Inhibitor ◽

Structural Basis ◽

Rna Dependent Rna Polymerase ◽

Structural Mechanism ◽

Template Strand ◽

Viral Rdrp ◽

Biochemical Assays ◽

Catalytic Active Site

Abstract The COVID-19 pandemic by non-stop infections of SARS-CoV-2 has continued to ravage many countries worldwide. Here we report the discovery of suramin, a 100-year-old drug, as a potent inhibitor of the SARS-CoV-2 RNA dependent RNA polymerase (RdRp) through blocking the binding of RNA to the enzyme. In biochemical assays, suramin and its derivatives are at least 20-fold more potent than remdesivir, the currently approved nucleotide drug for COVID-19. The 2.6 Å cryo-EM structure of the viral RdRp bound to suramin reveals two binding sites of suramin, with one site directly blocking the binding of the RNA template strand and the other site clash with the RNA primer strand near the RdRp catalytic active site. Furthermore, suramin potently inhibits SARS-CoV-2 duplication in Vero E6 cells. These results provide a structural mechanism for the first non-nucleotide inhibitor of the SARS-CoV-2 RdRp and a rationale for repurposing suramin for treating COVID-19.

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An ATR and CHK1 kinase signaling mechanism that limits origin firing during unperturbed DNA replication

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1903418116 ◽

2019 ◽

Vol 116 (27) ◽

pp. 13374-13383 ◽

Cited By ~ 26

Author(s):

Tatiana N. Moiseeva ◽

Yandong Yin ◽

Michael J. Calderon ◽

Chenao Qian ◽

Sandra Schamus-Haynes ◽

...

Keyword(s):

Dna Replication ◽

Kinase Activity ◽

Kinase Inhibitors ◽

S Phase ◽

Dna Lesions ◽

Dependent Manner ◽

Kinase Signaling ◽

Origin Firing ◽

Atr Kinase ◽

Chk1 Kinase

DNA damage-induced signaling by ATR and CHK1 inhibits DNA replication, stabilizes stalled and collapsed replication forks, and mediates the repair of multiple classes of DNA lesions. We and others have shown that ATR kinase inhibitors, three of which are currently undergoing clinical trials, induce excessive origin firing during unperturbed DNA replication, indicating that ATR kinase activity limits replication initiation in the absence of damage. However, the origins impacted and the underlying mechanism(s) have not been described. Here, we show that unperturbed DNA replication is associated with a low level of ATR and CHK1 kinase signaling and that inhibition of this signaling induces dormant origin firing at sites of ongoing replication throughout the S phase. We show that ATR and CHK1 kinase inhibitors induce RIF1 Ser2205 phosphorylation in a CDK1-dependent manner, which disrupts an interaction between RIF1 and PP1 phosphatase. Thus, ATR and CHK1 signaling suppresses CDK1 kinase activity throughout the S phase and stabilizes an interaction between RIF1 and PP1 in replicating cells. PP1 dephosphorylates key CDC7 and CDK2 kinase substrates to inhibit the assembly and activation of the replicative helicase. This mechanism limits origin firing during unperturbed DNA replication in human cells.

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Checkpoint regulation of replication forks: global or local?

Biochemical Society Transactions ◽

10.1042/bst20130197 ◽

2013 ◽

Vol 41 (6) ◽

pp. 1701-1705 ◽

Cited By ~ 8

Author(s):

Divya Ramalingam Iyer ◽

Nicholas Rhind

Keyword(s):

Dna Damage ◽

Replication Fork ◽

S Phase ◽

Cell Cycle Checkpoints ◽

Dna Damage Checkpoint ◽

Replication Forks ◽

Origin Firing ◽

Replication Fork Progression ◽

Late Origin ◽

Stalled Replication Forks

Cell-cycle checkpoints are generally global in nature: one unattached kinetochore prevents the segregation of all chromosomes; stalled replication forks inhibit late origin firing throughout the genome. A potential exception to this rule is the regulation of replication fork progression by the S-phase DNA damage checkpoint. In this case, it is possible that the checkpoint is global, and it slows all replication forks in the genome. However, it is also possible that the checkpoint acts locally at sites of DNA damage, and only slows those forks that encounter DNA damage. Whether the checkpoint regulates forks globally or locally has important mechanistic implications for how replication forks deal with damaged DNA during S-phase.

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Dpb11 Controls the Association between DNA Polymerases α and ɛ and the Autonomously Replicating Sequence Region of Budding Yeast

Molecular and Cellular Biology ◽

10.1128/mcb.20.8.2809-2817.2000 ◽

2000 ◽

Vol 20 (8) ◽

pp. 2809-2817 ◽

Cited By ~ 115

Author(s):

Hiroshi Masumoto ◽

Akio Sugino ◽

Hiroyuki Araki

Keyword(s):

Dna Polymerases ◽

S Phase ◽

Wild Type ◽

Chromosomal Dna ◽

Origin Firing ◽

Autonomously Replicating Sequence ◽

Sequence Region ◽

Late Origin ◽

Checkpoint Genes ◽

Primase Complex

ABSTRACT Dpb11 is required for chromosomal DNA replication and the S-phase checkpoint in Saccharomyces cerevisiae. Here, we report detection of a physical complex containing Dpb11 and DNA polymerase ɛ (Dpb11-Polɛ complex). During the S phase of the cell cycle, Dpb11 associated preferentially with DNA fragments containing autonomously replicating sequences (ARSs), at the same time as Polɛ associated with these fragments. Association of Dpb11 and Polɛ with these fragments was mutually dependent, suggesting that the Dpb11-Polɛ complex associates with the ARS. Moreover, Dpb11 was required for the association of Polα-primase with the fragments. Thus, it seems likely that association of the Dpb11-Polɛ complex with the ARS fragments is required for the association of the Polα-primase complex. Hydroxyurea inhibits late-origin firing in S. cerevisiae, and the checkpoint genes, RAD53 and MEC1, are involved in this inhibition. In the presence of hydroxyurea at temperatures permissive for cell growth, Polɛ in dpb11-1 cells associated with early- and late-origin fragments. In wild-type cells, however, it associated only with early-origin fragments. This indicates that Dpb11 may also be involved in the regulation of late-origin firing. Overall, these results suggest that Dpb11 controls the association between DNA polymerases α and ɛ and the ARS.

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Cryo-EM structure of VASH1-SVBP bound to microtubules

eLife ◽

10.7554/elife.58157 ◽

2020 ◽

Vol 9 ◽

Cited By ~ 2

Author(s):

Faxiang Li ◽

Yang Li ◽

Xuecheng Ye ◽

Haishan Gao ◽

Zhubing Shi ◽

...

Keyword(s):

Electron Microscopy ◽

Active Site ◽

Binding Protein ◽

Substrate Preference ◽

Structural Basis ◽

Globular Domain ◽

Specific Interactions ◽

Cryo Electron Microscopy ◽

Positively Charged

The dynamic tyrosination-detyrosination cycle of α-tubulin regulates microtubule functions. Perturbation of this cycle impairs mitosis, neural physiology, and cardiomyocyte contraction. The carboxypeptidases vasohibins 1 and 2 (VASH1 and VASH2), in complex with the small vasohibin-binding protein (SVBP), mediate α-tubulin detyrosination. These enzymes detyrosinate microtubules more efficiently than soluble αβ-tubulin heterodimers. The structural basis for this substrate preference is not understood. Using cryo-electron microscopy (cryo-EM), we have determined the structure of human VASH1-SVBP bound to microtubules. The acidic C-terminal tail of α-tubulin binds to a positively charged groove near the active site of VASH1. VASH1 forms multiple additional contacts with the globular domain of α-tubulin, including contacts with a second α-tubulin in an adjacent protofilament. Simultaneous engagement of two protofilaments by VASH1 can only occur within the microtubule lattice, but not with free αβ heterodimers. These lattice-specific interactions enable preferential detyrosination of microtubules by VASH1.

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Diminished S-Phase Cyclin-Dependent Kinase Function Elicits Vital Rad53-Dependent Checkpoint Responses in Saccharomyces cerevisiae

Molecular and Cellular Biology ◽

10.1128/mcb.24.23.10208-10222.2004 ◽

2004 ◽

Vol 24 (23) ◽

pp. 10208-10222 ◽

Cited By ~ 33

Author(s):

Daniel G. Gibson ◽

Jennifer G. Aparicio ◽

Fangfang Hu ◽

Oscar M. Aparicio

Keyword(s):

Saccharomyces Cerevisiae ◽

Dna Damage ◽

Dna Replication ◽

Replication Origin ◽

S Phase ◽

Replication Forks ◽

Cyclin Dependent Kinase ◽

Chromosomal Dna ◽

Origin Firing ◽

Late Origin

ABSTRACT Cyclin-dependent kinase (CDK) is required for the initiation of chromosomal DNA replication in eukaryotes. In Saccharomyces cerevisiae, the Clb5 and Clb6 cyclins activate Cdk1 and drive replication origin firing. Deletion of CLB5 reduces initiation of DNA synthesis from late-firing origins. We have examined whether checkpoints are activated by loss of Clb5 function and whether checkpoints are responsible for the DNA replication defects associated with loss of Clb5 function. We present evidence for activation of Rad53 and Ddc2 functions with characteristics suggesting the presence of DNA damage. Deficient late origin firing in clb5Δ cells is not due to checkpoint regulation, but instead, directly reflects the decreased abundance of S-phase CDK, as Clb6 activates late origins when its dosage is increased. Moreover, the viability of clb5Δ cells depends on Rad53. Activation of Rad53 by either Mrc1 or Rad9 contributes to the survival of clb5Δ cells, suggesting that both DNA replication and damage pathways are responsive to the decreased origin usage. These results suggest that reduced origin usage leads to stress or DNA damage at replication forks, necessitating the function of Rad53 in fork stabilization. Consistent with the notion that decreased S-CDK function creates stress at replication forks, deletion of RRM3 helicase, which facilitates replisome progression, greatly diminished the growth of clb5Δ cells. Together, our findings indicate that deregulation of S-CDK function has the potential to exacerbate genomic instability by reducing replication origin usage.

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Precocious S-Phase Entry in Budding Yeast Prolongs Replicative State and Increases Dependence Upon Rad53 for Viability

Genetics ◽

10.1093/genetics/160.1.123 ◽

2002 ◽

Vol 160 (1) ◽

pp. 123-136

Author(s):

Julia M Sidorova ◽

Linda L Breeden

Keyword(s):

Kinase Activity ◽

Growth Conditions ◽

S Phase ◽

Wild Type ◽

Origin Firing ◽

Phase Progression ◽

Rad53 Kinase ◽

Replication Intermediates ◽

Phase Entry ◽

In Cells

Abstract Precocious entry into S phase due to overproduction of G1 regulators can cause genomic instability. The mechanisms of this phenomenon are largely unknown. We explored the consequences of precocious S phase in yeast by overproducing a deregulated form of Swi4 (Swi4-t). Swi4 is a late G1-specific transcriptional activator that, in complex with Swi6, binds to SCB elements and activates late G1-specific genes, including G1 cyclins. We find that wild-type cells tolerate Swi4-t, whereas checkpoint-deficient rad53-11 cells lose viability within several divisions when Swi4-t is overproduced. Rad53 kinase activity is increased in cells overproducing Swi4-t, indicating activation of the checkpoint. We monitored the transition from G1 to S in cells with Swi4-t and found that there is precocious S-phase entry and that the length of S phase is extended. Moreover, there were more replication intermediates, and firing of at least a subset of origins may have been more extensive in the cells expressing Swi4-t. Our working hypothesis is that Rad53 modulates origin firing based upon growth conditions to optimize the rate of S-phase progression without adversely affecting fidelity. This regulation becomes essential when S phase is influenced by Swi4-t.

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The Function and Regulation of Budding Yeast Swe1 in Response to Interrupted DNA Synthesis

Molecular Biology of the Cell ◽

10.1091/mbc.e05-11-1093 ◽

2006 ◽

Vol 17 (6) ◽

pp. 2746-2756 ◽

Cited By ~ 32

Author(s):

Hong Liu ◽

Yanchang Wang

Keyword(s):

Dna Synthesis ◽

Budding Yeast ◽

S Phase ◽

Negative Regulator ◽

Basic Unit ◽

Origin Firing ◽

Protein Levels ◽

Late Origin ◽

S Phase Checkpoint

Periodically regulated cyclin-dependent kinase (Cdk) is required for DNA synthesis and mitosis. Hydroxyurea (HU) inhibits DNA synthesis by depleting dNTPs, the basic unit for DNA synthesis. HU treatment triggers the S-phase checkpoint, which arrests cells at S-phase, inhibits late origin firing and stabilizes replication forks. Using budding yeast as a model system, we found that Swe1, a negative regulator of Cdk, appears at S-phase and accumulates in HU treatment cells. Interestingly, this accumulation is not dependent on S-phase checkpoint. Δhsl1, Δhsl7, and cdc5-2 mutants, which have defects in Swe1 degradation, show HU sensitivity because of high Swe1 protein levels. We further demonstrated that their HU sensitivity is not a result of DNA damage accumulation or incomplete DNA synthesis; instead the sensitivity is due to their dramatically delayed recovery from HU-induced S-phase arrest. Strikingly, our in vivo data indicate that Swe1 inhibits the kinase activity of Clb2-Cdk1, but not that of Clb5-Cdk1. Therefore, S-phase accumulated Swe1 prevents Clb2-Cdk1–mediated mitotic activities, but has little effects on Clb5-Cdk1–associated S-phase progression.

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