scholarly journals Modes of Overinitiation, dnaA Gene Expression, and Inhibition of Cell Division in a Novel Cold-Sensitive hda Mutant of Escherichia coli

2008 ◽  
Vol 190 (15) ◽  
pp. 5368-5381 ◽  
Author(s):  
Kazuyuki Fujimitsu ◽  
Masayuki Su'etsugu ◽  
Yoko Yamaguchi ◽  
Kensaku Mazda ◽  
Nisi Fu ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Cell Division ◽  
Cell Cycle Progression ◽  
Synthetic Lethality ◽  
Active Form ◽  
Dependent Manner ◽  
Chromosomal Replication ◽  
Independent Manner ◽  
Multiple Copies ◽  
Cold Sensitive

ABSTRACT The chromosomal replication cycle is strictly coordinated with cell cycle progression in Escherichia coli. ATP-DnaA initiates replication, leading to loading of the DNA polymerase III holoenzyme. The DNA-loaded form of the β clamp subunit of the polymerase binds the Hda protein, which promotes ATP-DnaA hydrolysis, yielding inactive ADP-DnaA. This regulation is required to repress overinitiation. In this study, we have isolated a novel cold-sensitive hda mutant, the hda-185 mutant. The hda-185 mutant caused overinitiation of chromosomal replication at 25°C, which most likely led to blockage of replication fork progress. Consistently, the inhibition of colony formation at 25°C was suppressed by disruption of the diaA gene, an initiation stimulator. Disruption of the seqA gene, an initiation inhibitor, showed synthetic lethality with hda-185 even at 42°C. The cellular ATP-DnaA level was increased in an hda-185-dependent manner. The cellular concentrations of DnaA protein and dnaA mRNA were comparable at 25°C to those in a wild-type hda strain. We also found that multiple copies of the ribonucleotide reductase genes (nrdAB or nrdEF) or dnaB gene repressed overinitiation. The cellular levels of dATP and dCTP were elevated in cells bearing multiple copies of nrdAB. The catalytic site within NrdA was required for multicopy suppression, suggesting the importance of an active form of NrdA or elevated levels of deoxyribonucleotides in inhibition of overinitiation in the hda-185 cells. Cell division in the hda-185 mutant was inhibited at 25°C in a LexA regulon-independent manner, suggesting that overinitiation in the hda-185 mutant induced a unique division inhibition pathway.

Inactivation of YME1, a member of the ftsH-SEC18-PAS1-CDC48 family of putative ATPase-encoding genes, causes increased escape of DNA from mitochondria in Saccharomyces cerevisiae

1993 ◽  
Vol 13 (9) ◽  
pp. 5418-5426 ◽  
Author(s):  
P E Thorsness ◽  
K H White ◽  
T D Fox
Keyword(s):  
Escherichia Coli ◽  
Saccharomyces Cerevisiae ◽  
Cell Division ◽  
Synthetic Lethality ◽  
Nuclear Gene ◽  
Growth Defect ◽  
Glucose Medium ◽  
Cold Sensitive ◽  
Encoding Genes ◽  
Six Genes

The yeast nuclear gene YME1 was one of six genes recently identified in a screen for mutations that elevate the rate at which DNA escapes from mitochondria and migrates to the nucleus. yme1 mutations, including a deletion, cause four known recessive phenotypes: an elevation in the rate at which copies of TRP1 and ARS1, integrated into the mitochondrial genome, escape to the nucleus; a heat-sensitive respiratory-growth defect; a cold-sensitive growth defect on rich glucose medium; and synthetic lethality in rho- (cytoplasmic petite) cells. The cloned YME1 gene complements all of these phenotypes. The gene product, Yme1p, is immunologically detectable as an 82-kDa protein present in mitochondria. Yme1p is a member of a family of homologous putative ATPases, including Sec18p, Pas1p, Cdc48p, TBP-1, and the FtsH protein. Yme1p is most similar to the Escherichia coli FtsH protein, an essential protein involved in septum formation during cell division. This observation suggests the hypothesis that Yme1p may play a role in mitochondrial fusion and/or division.

scholarly journals Inactivation of YME1, a member of the ftsH-SEC18-PAS1-CDC48 family of putative ATPase-encoding genes, causes increased escape of DNA from mitochondria in Saccharomyces cerevisiae.

1993 ◽  
Vol 13 (9) ◽  
pp. 5418-5426 ◽  
Author(s):  
P E Thorsness ◽  
K H White ◽  
T D Fox
Keyword(s):  
Escherichia Coli ◽  
Saccharomyces Cerevisiae ◽  
Cell Division ◽  
Synthetic Lethality ◽  
Nuclear Gene ◽  
Growth Defect ◽  
Glucose Medium ◽  
Cold Sensitive ◽  
Encoding Genes ◽  
Six Genes

The yeast nuclear gene YME1 was one of six genes recently identified in a screen for mutations that elevate the rate at which DNA escapes from mitochondria and migrates to the nucleus. yme1 mutations, including a deletion, cause four known recessive phenotypes: an elevation in the rate at which copies of TRP1 and ARS1, integrated into the mitochondrial genome, escape to the nucleus; a heat-sensitive respiratory-growth defect; a cold-sensitive growth defect on rich glucose medium; and synthetic lethality in rho- (cytoplasmic petite) cells. The cloned YME1 gene complements all of these phenotypes. The gene product, Yme1p, is immunologically detectable as an 82-kDa protein present in mitochondria. Yme1p is a member of a family of homologous putative ATPases, including Sec18p, Pas1p, Cdc48p, TBP-1, and the FtsH protein. Yme1p is most similar to the Escherichia coli FtsH protein, an essential protein involved in septum formation during cell division. This observation suggests the hypothesis that Yme1p may play a role in mitochondrial fusion and/or division.

scholarly journals Characterization of YmgF, a 72-Residue Inner Membrane Protein That Associates with the Escherichia coli Cell Division Machinery

2008 ◽  
Vol 191 (1) ◽  
pp. 333-346 ◽  
Author(s):  
Gouzel Karimova ◽  
Carine Robichon ◽  
Daniel Ladant
Keyword(s):  
Escherichia Coli ◽  
Cell Division ◽  
Membrane Protein ◽  
Integral Membrane Protein ◽  
Dependent Manner ◽  
E Coli ◽  
Division Site ◽  
Inner Membrane Protein ◽  
Two Hybrid

ABSTRACT Formation of the Escherichia coli division septum is catalyzed by a number of essential proteins (named Fts) that assemble into a ring-like structure at the future division site. Many of these Fts proteins are intrinsic transmembrane proteins whose functions are largely unknown. In the present study, we attempted to identify a novel putative component(s) of the E. coli cell division machinery by searching for proteins that could interact with known Fts proteins. To do that, we used a bacterial two-hybrid system based on interaction-mediated reconstitution of a cyclic AMP (cAMP) signaling cascade to perform a library screening in order to find putative partners of E. coli cell division protein FtsL. Here we report the characterization of YmgF, a 72-residue integral membrane protein of unknown function that was found to associate with many E. coli cell division proteins and to localize to the E. coli division septum in an FtsZ-, FtsA-, FtsQ-, and FtsN-dependent manner. Although YmgF was previously shown to be not essential for cell viability, we found that when overexpressed, YmgF was able to overcome the thermosensitive phenotype of the ftsQ1(Ts) mutation and restore its viability under low-osmolarity conditions. Our results suggest that YmgF might be a novel component of the E. coli cell division machinery.

The Jak2V617F Oncogene Associated with Polycythemia Vera Regulates G1/S-Phase Transition.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 3510-3510
Author(s):  
Martin Sattler ◽  
Christoph Walz ◽  
Brian J. Crowley ◽  
Jessica L. Gramlich ◽  
Kendra L. King ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cell Growth ◽  
Polycythemia Vera ◽  
Cell Cycle Progression ◽  
Kinase Inhibitor ◽  
Active Form ◽  
Leukemia Cell ◽  
Leukemia Cell Line ◽  
Dependent Manner ◽  
Hel Cells

Abstract The V617F activating point mutation in Jak2 has recently been detected in a high proportion of patients with the myeloproliferative disorders polycythemia vera, essential thrombocythemia, and idiopathic myelofibrosis. Using the Jak2V617F-mutant erythroid leukemia cell line HEL as a model, potential mechanisms that contribute to transformation were investigated. Inhibition of Jak2V617F with a small molecule kinase inhibitor reduced cell growth of HEL cells in a dose dependent manner with an IC50 of 300 nM. This inhibition of growth was associated with a G1 cell cycle arrest, with minimal or delayed apoptosis. The major Jak2 target in normal hematopoietic cells, STAT5, was found to be activated by Jak2V617F. Treatment of the cells with either a Jak2 kinase inhibitor, or with a Jak2-targeted siRNA, decreased STAT5 activation, and also resulted in decreased expression of cyclin D2 and increased expression of p27Kip. Of interest, we found that Jak2V617F induced high levels of reactive oxygen species (ROS), an activity associated with several other tyrosine kinase oncogenes. Expression of a constitutively active form of STAT5 by itself was capable reducing expression of p27Kip and increasing production of ROS, suggesting that each of these signaling events are downstream of STAT5. Additionally, treatment of HEL cells with the anti-oxidant N-acetylcystein increased expression of p27Kip, suggesting that Jak2V617F regulates cell cycle progression at least in part through STAT5 activation of ROS, and ROS regulation of p27Kip. Cell growth of HEL cells was found to be blocked by anti-oxidants. Overall, our results suggest that constitutive activation of Jak2 contributes to a transforming phenotype and therefore hints at novel targets for drug development that may aid traditional therapy.

scholarly journals Cyclin F-Chk1 synthetic lethality mediated by E2F1 degradation

2019 ◽  
Author(s):  
Kamila Burdova ◽  
Hongbin Yang ◽  
Roberta Faedda ◽  
Samuel Hume ◽  
Daniel Ebner ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Dna Replication ◽  
Cell Cycle Progression ◽  
Kinase Inhibitors ◽  
Synthetic Lethality ◽  
Checkpoint Inhibitors ◽  
Dependent Manner ◽  
Cycle Progression ◽  
Multiple Substrates ◽  
Cyclin F

SummaryCyclins are central engines of cell cycle progression when partnered with Cyclin Dependent Kinases (CDKs). Among the different cyclins controlling cell cycle progression, cyclin F does not partner with a CDK, but forms an E3 ubiquitin ligase, assembling through the F-box domain, an Skp1-Cul1-F-box (SCF) module. Although multiple substrates of cyclin F have been identified the vulnerabilities of cells lacking cyclin F are not known. Thus, we assessed viability of cells lacking cyclin F upon challenging cells with more than 200 kinase inhibitors. The screen revealed a striking synthetic lethality between Chk1 inhibition and cyclin F loss. Chk1 inhibition in cells lacking cyclin F leads to DNA replication catastrophe. The DNA replication catastrophe depends on the accumulation of E2F1 in cyclin F depleted cells. We observe that SCFcyclin F promotes E2F1 degradation after Chk1 inhibitors in a CDK dependent manner. Thus, Cyclin F restricts E2F1 activity during cell cycle and upon checkpoint inhibition to prevent DNA replication stress. Our findings pave the way for patient selection in the clinical use of checkpoint inhibitors.

scholarly journals Casein Kinase II is Required for Proper Cell Division and Acts as a Negative Regulator of Centrosome Duplication in C. elegans Embryos

2016 ◽  
Author(s):  
Jeffrey C. Medley ◽  
Megan M. Kabara ◽  
Michael D. Stubenvoll ◽  
Lauren E. DeMeyer ◽  
Mi Hye Song
Keyword(s):  
Cell Cycle ◽  
Cell Division ◽  
Cell Cycle Progression ◽  
Casein Kinase Ii ◽  
Casein Kinase ◽  
Negative Regulator ◽  
Centrosome Duplication ◽  
Dependent Manner ◽  
C Elegans ◽  
Mother Centriole

Summary statementThe conserved protein kinase CK2 negatively regulates centrosome assembly and is required for proper cell cycle progression and cytokinesis in early C. elegans embryos.AbstractCentrosomes are the primary microtubule-organizing centers that orchestrate microtubule dynamics during the cell cycle. The correct number of centrosomes is pivotal for establishing bipolar mitotic spindles that ensure accurate segregation of chromosomes. Thus, centrioles must duplicate once per cell cycle, one daughter per mother centriole, the process of which requires highly coordinated actions among core factors and modulators. Protein phosphorylation is shown to regulate the stability, localization and activity of centrosome proteins. Here, we report the function of Casein Kinase II (CK2) in early C. elegans embryos. The catalytic subunit (KIN-3/CK2α) of CK2 localizes to nuclei, centrosomes and midbodies. Inactivating CK2 leads to cell division defects, including chromosome missegregation, cytokinesis failure and aberrant centrosome behavior. Furthermore, depletion or inhibiting kinase activity of CK2 results in elevated ZYG-1 levels at centrosomes, restoring centrosome duplication and embryonic viability to zyg-1 mutants. Our data suggest that CK2 functions in cell division and negatively regulates centrosome duplication in a kinase-dependent manner.

scholarly journals Contribution of CAF-I to Anaphase-Promoting-Complex-Mediated Mitotic Chromatin Assembly in Saccharomyces cerevisiae

2005 ◽  
Vol 4 (4) ◽  
pp. 673-684 ◽  
Author(s):  
Troy A. A. Harkness ◽  
Terra G. Arnason ◽  
Charmaine Legrand ◽  
Marnie G. Pisclevich ◽  
Gerald F. Davies ◽  
...  
Keyword(s):  
Chromatin Assembly ◽  
Dependent Manner ◽  
Anaphase Promoting Complex ◽  
Mitotic Progression ◽  
Independent Manner ◽  
Expression Of Genes ◽  
Genes Encoding ◽  
Multiple Copies ◽  
The Individual ◽  
Mitotic Chromatin

ABSTRACT The anaphase-promoting complex (APC) is required for mitotic progression and genomic stability. Recently, we demonstrated that the APC is also required for mitotic chromatin assembly and longevity. Here, we investigated the role the APC plays in chromatin assembly. We show that apc5 CA mutations genetically interact with the CAF-I genes as well as ASF1, HIR1, and HIR2. When present in multiple copies, the individual CAF-I genes, CAC1, CAC2, and MSI1, suppress apc5 CA phenotypes in a CAF-1- and Asf1p-independent manner. CAF-I and the APC functionally overlap, as cac1Δ cac2Δ msi1Δ (caf1Δ) cells expressing apc5 CA exhibit a phenotype more severe than that of apc5 CA or caf1Δ. The Ts− phenotypes observed in apc5 CA and apc5 CA caf mutants may be rooted in compromised histone metabolism, as coexpression of histones H3 and H4 suppressed the Ts− defects. Synthetic genetic interactions were also observed in apc5 CA asf1Δ cells. Furthermore, increased expression of genes encoding Asf1p, Hir1p, and Hir2p suppressed the apc5 CA Ts− defect in a CAF-I-dependent manner. Together, these results suggest the existence of a complex molecular mechanism controlling APC-dependent chromatin assembly. Our data suggest the APC functions with the individual CAF-I subunits, Asf1p, and the Hir1p and Hir2p proteins. However, Asf1p and an intact CAF-I complex are dispensable for CAF-I subunit suppression, whereas CAF-I is necessary for ASF1, HIR1, and HIR2 suppression of apc5 CA phenotypes. We discuss the implications of our observations.

scholarly journals Comparative Gene Expression Profiles Following UV Exposure in Wild-Type and SOS-Deficient Escherichia coli

Genetics ◽  
2001 ◽  
Vol 158 (1) ◽  
pp. 41-64 ◽  
Author(s):  
Justin Courcelle ◽  
Arkady Khodursky ◽  
Brian Peter ◽  
Patrick O Brown ◽  
Philip C Hanawalt
Keyword(s):  
Gene Expression ◽  
Escherichia Coli ◽  
Uv Irradiation ◽  
Expression Profiles ◽  
Gene Expression Profiles ◽  
Uv Exposure ◽  
Dependent Manner ◽  
Wild Type ◽  
Independent Manner ◽  
E Coli

Abstract The SOS response in UV-irradiated Escherichia coli includes the upregulation of several dozen genes that are negatively regulated by the LexA repressor. Using DNA microarrays containing amplified DNA fragments from 95.5% of all open reading frames identified on the E. coli chromosome, we have examined the changes in gene expression following UV exposure in both wild-type cells and lexA1 mutants, which are unable to induce genes under LexA control. We report here the time courses of expression of the genes surrounding the 26 documented lexA-regulated regions on the E. coli chromosome. We observed 17 additional sites that responded in a lexA-dependent manner and a large number of genes that were upregulated in a lexA-independent manner although upregulation in this manner was generally not more than twofold. In addition, several transcripts were either downregulated or degraded following UV irradiation. These newly identified UV-responsive genes are discussed with respect to their possible roles in cellular recovery following exposure to UV irradiation.

scholarly journals Fragile X–Related Protein 1 Regulates Nucleoporin Localization in a Cell Cycle–Dependent Manner

2021 ◽  
Vol 9 ◽  
Author(s):  
Arantxa Agote-Arán ◽  
Junyan Lin ◽  
Izabela Sumara
Keyword(s):  
Cell Cycle ◽  
Mammalian Cells ◽  
Cell Cycle Progression ◽  
Fragile X ◽  
Protein Translation ◽  
Nuclear Pore ◽  
Nuclear Pore Complexes ◽  
Dependent Manner ◽  
Independent Manner ◽  
Cycle Dependent

Nuclear pore complexes (NPCs) are embedded in the nuclear envelope (NE) where they ensure the transport of macromolecules between the nucleus and the cytoplasm. NPCs are built from nucleoporins (Nups) through a sequential assembly order taking place at two different stages during the cell cycle of mammalian cells: at the end of mitosis and during interphase. In addition, fragile X–related proteins (FXRPs) can interact with several cytoplasmic Nups and facilitate their localization to the NE during interphase likely through a microtubule-dependent mechanism. In the absence of FXRPs or microtubule-based transport, Nups aberrantly localize to the cytoplasm forming the so-called cytoplasmic nucleoporin granules (CNGs), compromising NPCs’ function on protein export. However, it remains unknown if Nup synthesis or degradation mechanisms are linked to the FXRP–Nup pathway and if and how the action of FXRPs on Nups is coordinated with the cell cycle progression. Here, we show that Nup localization defects observed in the absence of FXR1 are independent of active protein translation. CNGs are cleared in an autophagy- and proteasome-independent manner, and their presence is restricted to the early G1 phase of the cell cycle. Our results thus suggest that a pool of cytoplasmic Nups exists that contributes to the NPC assembly specifically during early G1 to ensure NPC homeostasis at a short transition from mitosis to the onset of interphase.

scholarly journals Acquired Resistance of Escherichia coli to Complement Lysis by Binding of Glycophosphoinositol-Anchored Protectin (CD59)

1998 ◽  
Vol 66 (5) ◽  
pp. 1928-1933 ◽  
Author(s):  
Riina Rautemaa ◽  
Gary A. Jarvis ◽  
Pertti Marnila ◽  
Seppo Meri
Keyword(s):  
Escherichia Coli ◽  
Cell Membranes ◽  
Active Form ◽  
Acquired Resistance ◽  
Membrane Attack Complex ◽  
Green Light ◽  
Soluble Form ◽  
Enzyme Linked Immunosorbent Assay ◽  
Dependent Manner ◽  
Complement Lysis

ABSTRACT Protectin (CD59) is a glycophosphoinsitol (GPI)-anchored defender of human cells against lysis by the membrane attack complex of complement. In this study, we examined whether protectin released from human cell membranes can incorporate into the surface of gram-negative bacteria. Analysis by using radiolabeled protectin, immunofluorescence, flow cytometry, and whole-cell enzyme-linked immunosorbent assay demonstrated that protectin bound to nonencapsulated Escherichia coli EH237 (Re) and EH234 (Ra) in a calcium-dependent manner. The incorporation required the GPI-phospholipid moiety since no binding of a phospholipid-free soluble form of protectin was observed. Mg2+ did not enhance the binding, and a polysialic acid capsule prevented it (strain IH3080 [O18:K1:H8]). Bound protectin inhibited the C5b-9 neoantigen expression on complement-treated bacteria. Protection against complement lysis was observed in both a colony counting assay and a bioluminescence assay, where viable EH234 bacteria expressing the luciferase gene emitted green light in the presence of the luciferine substrate. In general, two- to four-times-higher serum concentrations were needed to obtain 50% lysis of protectin-coated versus noncoated bacteria. The results indicate that protectin can incorporate in a functionally active form into the cell membranes of the two nonencapsulated deep rough E. coli strains studied.

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