scholarly journals Rad51 catalytic mutants differentially affect the Rad51 nucleoprotein filament in vivo

2016 ◽  
Author(s):  
Maureen M. Mundia ◽  
Alissa C. Magwood ◽  
Mark D. Baker
Keyword(s):  
Homologous Recombination ◽  
Dna Synthesis ◽  
Cell Lines ◽  
Atp Hydrolysis ◽  
Dominant Negative ◽  
Strand Exchange ◽  
Wild Type ◽  
Hybridoma Cell Lines ◽  
Insight Into

ABSTRACTIn this study, we utilized mouse hybridoma cell lines stably expressing ectopic wild-type Rad51, or the Rad51-K133A and Rad51-K133R catalytic mutants deficient in ATP binding and ATP hydrolysis, respectively, to investigate effects on the Rad51 nucleoprotein filament in vivo. Immunoprecipitation studies reveal interactions between ectopic wild-type Rad51, Rad51-K133A and Rad51-K133R and endogenous Rad51, Brca2 and p53 proteins. Importantly, the expression of Rad51-K133A and Rad51-K133R catalytic mutants (but not wild-type Rad51) targets endogenous Rad51, Brca2 and p53 proteins for proteasome-mediated degradation. Expression of Rad51-K133R significantly reduces nascent DNA synthesis (3’ polymerization) during homologous recombination (HR), but the effects of Rad51-K133A on 3’ polymerization are considerably more severe. Provision of additional wild-type Rad51 in cell lines expressing Rad51-K133A or Rad51-K133R does not restore diminished levels of endogenous Brca2, Rad51 or p53, nor restore the deficiency in 3’ polymerization. Cells expressing Rad51-K133A are also significantly reduced in their capacity to drive strand exchange through regions of heterology. Our results reveal an interesting mechanistic dichotomy in the way mutant Rad51-K133A and Rad51-K133R proteins influence 3’ polymerization and provide novel insight into the mechanism of their dominant-negative phenotypes.

scholarly journals Phenotypic reversions at the W/Kit locus mediated by mitotic recombination in mice.

1995 ◽  
Vol 15 (11) ◽  
pp. 5898-5905 ◽  
Author(s):  
P De Sepulveda ◽  
J L Guenet ◽  
J J Panthier
Keyword(s):  
Cell Lines ◽  
Receptor Tyrosine Kinase ◽  
Coat Color ◽  
Mitotic Recombination ◽  
Dominant Negative ◽  
Wild Type ◽  
Endogenous Expression ◽  
Complete Failure

The mouse W locus encodes Kit, the receptor tyrosine kinase for stem cell factor (SCF). Kit is required for several developmental processes, including the proliferation and survival of melanoblasts. Because of the nearly complete failure of Wrio/+ melanoblasts to colonize the skin, the costs of Wrio/+ mice are characterized by a majority of white hairs interspersed among pigmented hairs, giving a roan effect. However, 3.6% of Wrio/+ mice exhibit phenotypic reversions, i.e., spots of wild-type color on their coats with an otherwise mutant phenotype. Melanocyte cell lines were derived from each of six independent reversion spots on the skin of (C57BL/6 x DBA/2)F1 Wrio/+ mice. All six melanocyte cell lines exhibited the general characteristics common to normal, nonimmortal mouse melanocytes. Of these, three revertant cell lines had lost the dominant-negative Wrio allele following mitotic recombination between the centromere and the W locus. One of the cell lines remained Wrio/+ but showed (i) stimulation in response to SCF and (ii) increased Kit expression, suggesting that the Wrio mutation can be rescued by increased endogenous expression of the c-kit proto-oncogene. Finally, two cell lines showed no detectable genetic change at the W/Kit locus and failed to respond to SCF stimulation in vitro. These results demonstrate that mitotic recombination can create large patches of wild-type hair on the coats of Wrio/+ mutant mice. This shows that mitotic recombination occurs spontaneously in normal healthy tissue in vivo. Moreover, these experiments confirm that other mechanisms, not associated with loss of heterozygosity, may account for the coat color reversion phenotype.

scholarly journals The Anti-Tumor Activity of the NEDD8 Inhibitor Pevonedistat in Neuroblastoma

2021 ◽  
Vol 22 (12) ◽  
pp. 6565
Author(s):  
Jennifer H. Foster ◽  
Eveline Barbieri ◽  
Linna Zhang ◽  
Kathleen A. Scorsone ◽  
Myrthala Moreno-Smith ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cell Lines ◽  
Neuroblastoma Cell ◽  
Wild Type ◽  
Tumor Weight ◽  
Cycle Arrest ◽  
P53 Status ◽  
Neuroblastoma Cell Lines ◽  
Anti Tumor Activity

Pevonedistat is a neddylation inhibitor that blocks proteasomal degradation of cullin–RING ligase (CRL) proteins involved in the degradation of short-lived regulatory proteins, including those involved with cell-cycle regulation. We determined the sensitivity and mechanism of action of pevonedistat cytotoxicity in neuroblastoma. Pevonedistat cytotoxicity was assessed using cell viability assays and apoptosis. We examined mechanisms of action using flow cytometry, bromodeoxyuridine (BrDU) and immunoblots. Orthotopic mouse xenografts of human neuroblastoma were generated to assess in vivo anti-tumor activity. Neuroblastoma cell lines were very sensitive to pevonedistat (IC50 136–400 nM). The mechanism of pevonedistat cytotoxicity depended on p53 status. Neuroblastoma cells with mutant (p53MUT) or reduced levels of wild-type p53 (p53si-p53) underwent G2-M cell-cycle arrest with rereplication, whereas p53 wild-type (p53WT) cell lines underwent G0-G1 cell-cycle arrest and apoptosis. In orthotopic neuroblastoma models, pevonedistat decreased tumor weight independent of p53 status. Control mice had an average tumor weight of 1.6 mg + 0.8 mg versus 0.5 mg + 0.4 mg (p < 0.05) in mice treated with pevonedistat. The mechanism of action of pevonedistat in neuroblastoma cell lines in vitro appears p53 dependent. However, in vivo studies using mouse neuroblastoma orthotopic models showed a significant decrease in tumor weight following pevonedistat treatment independent of the p53 status. Novel chemotherapy agents, such as the NEDD8-activating enzyme (NAE) inhibitor pevonedistat, deserve further study in the treatment of neuroblastoma.

scholarly journals RuvAB and RecG Are Not Essential for the Recovery of DNA Synthesis Following UV-Induced DNA Damage in Escherichia coli

Genetics ◽  
2004 ◽  
Vol 166 (4) ◽  
pp. 1631-1640 ◽  
Author(s):  
Janet R Donaldson ◽  
Charmain T Courcelle ◽  
Justin Courcelle
Keyword(s):  
Dna Damage ◽  
Dna Synthesis ◽  
Dna Lesions ◽  
Replication Forks ◽  
Wild Type ◽  
Replication Machinery ◽  
Dna Junctions ◽  
Fork Regression

Abstract Ultraviolet light induces DNA lesions that block the progression of the replication machinery. Several models speculate that the resumption of replication following disruption by UV-induced DNA damage requires regression of the nascent DNA or migration of the replication machinery away from the blocking lesion to allow repair or bypass of the lesion to occur. Both RuvAB and RecG catalyze branch migration of three- and four-stranded DNA junctions in vitro and are proposed to catalyze fork regression in vivo. To examine this possibility, we characterized the recovery of DNA synthesis in ruvAB and recG mutants. We found that in the absence of either RecG or RuvAB, arrested replication forks are maintained and DNA synthesis is resumed with kinetics that are similar to those in wild-type cells. The data presented here indicate that RecG- or RuvAB-catalyzed fork regression is not essential for DNA synthesis to resume following arrest by UV-induced DNA damage in vivo.

scholarly journals Chi-Dependent Intramolecular Recombination in Escherichia coli

Genetics ◽  
1998 ◽  
Vol 148 (2) ◽  
pp. 545-557
Author(s):  
Rachel Friedman-Ohana ◽  
Iris Karunker ◽  
Amikam Cohen
Keyword(s):  
Escherichia Coli ◽  
Homologous Recombination ◽  
Experimental System ◽  
Cis Acting ◽  
Enzymes Activity ◽  
Intramolecular Recombination ◽  
Insight Into

Abstract Homologous recombination in Escherichia coli is enhanced by a cis-acting octamer sequence named Chi (5′-GCTGGTGG-3′) that interacts with RecBCD. To gain insight into the mechanism of Chi-enhanced recombination, we recruited an experimental system that permits physical monitoring of intramolecular recombination by linear substrates released by in vivo restriction from infecting chimera phage. Recombination of the released substrates depended on recA, recBCD and cis-acting Chi octamers. Recombination proficiency was lowered by a xonA mutation and by mutations that inactivated the RuvABC and RecG resolution enzymes. Activity of Chi sites was influenced by their locations and by the number of Chi octamers at each site. A single Chi site stimulated recombination, but a combination of Chi sites on the two homologs was synergistic. These data suggest a role for Chi at both ends of the linear substrate. Chi was lost in all recombinational exchanges stimulated by a single Chi site. Exchanges in substrates with Chi sites on both homologs occurred in the interval between the sites as well as in the flanking interval. These observations suggest that the generation of circular products by intramolecular recombination involves Chi-dependent processing of one end by RecBCD and pairing of the processed end with its duplex homolog.

scholarly journals Overproduction of SecA Suppresses the Export Defect Caused by a Mutation in the Gene Encoding the Escherichia coli Export Chaperone SecB

1999 ◽  
Vol 181 (10) ◽  
pp. 3010-3017 ◽  
Author(s):  
Heather A. Cook ◽  
Carol A. Kumamoto
Keyword(s):  
Escherichia Coli ◽  
Outer Membrane Proteins ◽  
Protein A ◽  
In Vivo Studies ◽  
Wild Type ◽  
Gene Encoding ◽  
Precursor Proteins ◽  
Additional Support ◽  
Insight Into

ABSTRACT SecB is a cytosolic protein required for rapid and efficient export of particular periplasmic and outer membrane proteins inEscherichia coli. SecB promotes export by stabilizing newly synthesized precursor proteins in a nonnative conformation and by targeting the precursors to the inner membrane. Biochemical studies suggest that SecB facilitates precursor targeting by binding to the SecA protein, a component of the membrane-embedded translocation apparatus. To gain more insight into the functional interaction of SecB and SecA, in vivo, mutations in the secA locus that compensate for the export defect caused by the secBmissense mutation secBL75Q were isolated. Two suppressors were isolated, both of which led to the overproduction of wild-type SecA protein. In vivo studies demonstrated that the SecBL75Q mutant protein releases precursor proteins at a lower rate than does wild-type SecB. Increasing the level of SecA protein in the cell was found to reverse this slow-release defect, indicating that overproduction of SecA stimulates the turnover of SecBL75Q-precursor complexes. These findings lend additional support to the proposed pathway for precursor targeting in which SecB promotes targeting to the translocation apparatus by binding to the SecA protein.

Abstract 470: Recording Cell Migration Dynamics in Mouse Tissues With Cells Secreting Fluorescence Protein-tagged Collagen

2020 ◽  
Vol 127 (Suppl_1) ◽  
Author(s):  
Zhongming Chen
Keyword(s):  
Cell Migration ◽  
Cell Lines ◽  
Wild Type Mouse ◽  
Cardiac Progenitor Cells ◽  
Wild Type ◽  
Mouse Tissues ◽  
Migration Dynamics ◽  
Fluorescence Protein

Background: Cell migration is an important step involved in heart regeneration and many cardiovascular diseases. However, cell migration dynamics in vivo is poorly understood due to the challenges from mammal hearts, which are opaque and fast beating, and thus individual cardiac cells cannot be imaged or tracked. Aims: In this study, cell migration dynamics in the heart is recorded with a novel strategy, in which fluorescence protein-tagged collagen is secreted from cells and deposited into extracellular matrix, forming visible trails when cells are moving in tissues. As a proof-of-concept, transplanted migration dynamics of cardiac progenitor cells in mouse hearts were investaged. Methods: Stable cell lines expressing mCherry-tagged type I collagen were generated from isolated cardiac progenitor cells, ABCG2 + CD45 - CD31 - cells (side populations), or c-kit + CD45 - CD31 - cells (c-kit + CPCs). The cell migration dynamics were monitored and measured based on the cell trails after cell transplantation into mouse tissues. Results: The stable cell lines form red cell trails both in vitro and in vivo (Fig. 1A & 1B, Green: GFP; Red: mCherry-collagen I, Blue: DAPI, bar: 50 microns). In culture dishes, the cells form visible cell trails of fluorescence protein. The cell moving directions are random, with a speed of 288 +/- 79 microns/day (side populations, n=3) or 143 +/-37 microns/day (c-kit + CPCs, n=3). After transplantation into wild-type mouse hearts, the cells form highly tortuous trails along the gaps between the heart muscle fibers. Angle between a cell trail and a muscle fiber is 16+/-16 degree (n=3). Side populations migrate twice as fast as c-kit+ CPCs in the heart (16.0 +/-8.7 microns/day vs. 8.1+/-0.0 microns/day, n=3, respectively), 18 time slower than the respective speeds in vitro . Additionally, side populations migrate significantly faster in the heart than in the skeletal muscles (26.4+/-5.8 microns/day, n=3). The side populations move significantly faster in immunodeficient mouse hearts (36.7+/-13.3 microns/day, n=3, typically used for studying cell therapies) than in wild-type mouse hearts. Conclusion: For the first time, cell migration dynamics in living hearts is monitored and examined with genetically modified cell lines. This study may greatly advance the fields of cardiovascular biology.

scholarly journals Protein kinase D inhibits plasma membrane Na+/H+exchanger activity

1999 ◽  
Vol 277 (6) ◽  
pp. C1202-C1209 ◽  
Author(s):  
Robert S. Haworth ◽  
James Sinnett-Smith ◽  
Enrique Rozengurt ◽  
Metin Avkiran
Keyword(s):  
Plasma Membrane ◽  
Protein Kinase ◽  
Phorbol Ester ◽  
Fluorescent Protein ◽  
Dominant Negative ◽  
Protein Kinase D ◽  
Wild Type ◽  
Ph Indicator

The regulation of plasma membrane Na+/H+exchanger (NHE) activity by protein kinase D (PKD), a novel protein kinase C- and phorbol ester-regulated kinase, was investigated. To determine the effect of PKD on NHE activity in vivo, intracellular pH (pHi) measurements were made in COS-7 cells by microepifluorescence using the pH indicator cSNARF-1. Cells were transfected with empty vector (control), wild-type PKD, or its kinase-deficient mutant PKD-K618M, together with green fluorescent protein (GFP). NHE activity, as reflected by the rate of acid efflux ( J H), was determined in single GFP-positive cells following intracellular acidification. Overexpression of wild-type PKD had no significant effect on J H(3.48 ± 0.25 vs. 3.78 ± 0.24 mM/min in control at pHi 7.0). In contrast, overexpression of PKD-K618M increased J H (5.31 ± 0.57 mM/min at pHi 7.0; P < 0.05 vs. control). Transfection with these constructs produced similar effects also in A-10 cells, indicating that native PKD may have an inhibitory effect on NHE in both cell types, which is relieved by a dominant-negative action of PKD-K618M. Exposure of COS-7 cells to phorbol ester significantly increased J H in control cells but failed to do so in cells overexpressing either wild-type PKD (due to inhibition by the overexpressed PKD) or PKD-K618M (because basal J Hwas already near maximal). A fusion protein containing the cytosolic regulatory domain (amino acids 637–815) of NHE1 (the ubiquitous NHE isoform) was phosphorylated in vitro by wild-type PKD, but with low stoichiometry. These data suggest that PKD inhibits NHE activity, probably through an indirect mechanism, and represents a novel pathway in the regulation of the exchanger.

scholarly journals N- and O-glycosylation muteins of recombinant human erythropoietin secreted from BHK-21 cells

Blood ◽  
1995 ◽  
Vol 85 (5) ◽  
pp. 1229-1236 ◽  
Author(s):  
MR Fibi ◽  
P Hermentin ◽  
JU Pauly ◽  
L Lauffer ◽  
G Zettlmeissl
Keyword(s):  
Transient Expression ◽  
Cell Lines ◽  
Recombinant Human Erythropoietin ◽  
Biologically Active ◽  
Wild Type ◽  
Expression Levels ◽  
Human Erythropoietin ◽  
Culture Supernatants ◽  
Secretion Rates

Single-site glycomuteins of recombinant human erythropoietin (rhuEpo) were constructed and transiently and stably expressed in BHK-21 cells. The transient expression levels varied among muteins, being highest for mutein rhuEpoGln24 followed by wild-type rhuEpo (rhuEpowt). All other glycomuteins, including rhuEpoGln38, rhuEpoGln83, rhuEpoThr126, and rhuEpoGly126, were secreted at lower levels than rhuEpowt. Muteins expressed in stable cell lines showed similar differences in expression levels. Also each mutein could be affinity-purified from culture supernatants, and was biologically active in vivo. Based on secretion rates from BHK-21 cells, the most potent erythropoietin was rhuEpoGln24. This mutein is also considered to have biologic activities that are superior to rhuEpowt.

scholarly journals Enterococcus faecalis 6-Phosphogluconolactonase Is Required for Both Commensal and Pathogenic Interactions with Manduca sexta

2014 ◽  
Vol 83 (1) ◽  
pp. 396-404 ◽  
Author(s):  
Jonathan F. Holt ◽  
Megan R. Kiedrowski ◽  
Kristi L. Frank ◽  
Jing Du ◽  
Changhui Guan ◽  
...  
Keyword(s):  
Enterococcus Faecalis ◽  
Manduca Sexta ◽  
Wild Type ◽  
Genetic Determinants ◽  
Content Type ◽  
Polystyrene Plate ◽  
Insect Gut ◽  
Insight Into

Enterococcus faecalisis a commensal and pathogen of humans and insects. InManduca sexta,E. faecalisis an infrequent member of the commensal gut community, but its translocation to the hemocoel results in a commensal-to-pathogen switch. To investigateE. faecalisfactors required for commensalism, we identifiedE. faecalisgenes that are upregulated in the gut ofM. sextausing recombinase-basedin vivoexpression technology (RIVET). The RIVET screen produced 113 clones, from which we identified 50 genes that are more highly expressed in the insect gut than in culture. The most frequently recovered gene was locus OG1RF_11582, which encodes a 6-phosphogluconolactonase that we designatedpglA. ApglAdeletion mutant was impaired in both pathogenesis and gut persistence inM. sextaand produced enhanced biofilms compared with the wild type in anin vitropolystyrene plate assay. Mutation of four other genes identified by RIVET did not affect persistence in caterpillar guts but led to impaired pathogenesis. This is the first identification of genetic determinants forE. faecaliscommensal and pathogenic interactions withM. sexta. Bacterial factors identified in this model system may provide insight into colonization or persistence in other host-associated microbial communities and represent potential targets for interventions to preventE. faecalisinfections.

scholarly journals Norovirus Polymerase Fidelity Contributes to Viral Transmission In Vivo

mSphere ◽  
2016 ◽  
Vol 1 (5) ◽  
Author(s):  
Armando Arias ◽  
Lucy Thorne ◽  
Elsa Ghurburrun ◽  
Dalan Bailey ◽  
Ian Goodfellow
Keyword(s):  
Genetic Diversity ◽  
Virus Transmission ◽  
Viral Pathogenesis ◽  
High Fidelity ◽  
Wild Type ◽  
Replication Fidelity ◽  
Polymerase Fidelity ◽  
Natural Hosts ◽  
Insight Into

ABSTRACT Virus replication fidelity and hence the intrahost genetic diversity of viral populations are known to be intricately linked to viral pathogenesis and tropism as well as to immune and antiviral escape during infection. In this study, we investigated whether changes in replication fidelity can impact the ability of a virus to transmit between susceptible hosts by the use of a mouse model for norovirus. We show that a variant encoding a high-fidelity polymerase is transmitted less efficiently between mice than the wild-type strain. This constitutes the first experimental demonstration that the polymerase fidelity of viruses can impact transmission of infection in their natural hosts. These results provide further insight into potential reasons for the global emergence of pandemic human noroviruses that display alterations in the replication fidelity of their polymerases compared to nonpandemic strains. Intrahost genetic diversity and replication error rates are intricately linked to RNA virus pathogenesis, with alterations in viral polymerase fidelity typically leading to attenuation during infections in vivo. We have previously shown that norovirus intrahost genetic diversity also influences viral pathogenesis using the murine norovirus model, as increasing viral mutation frequency using a mutagenic nucleoside resulted in clearance of a persistent infection in mice. Given the role of replication fidelity and genetic diversity in pathogenesis, we have now investigated whether polymerase fidelity can also impact virus transmission between susceptible hosts. We have identified a high-fidelity norovirus RNA-dependent RNA polymerase mutant (I391L) which displays delayed replication kinetics in vivo but not in cell culture. The I391L polymerase mutant also exhibited lower transmission rates between susceptible hosts than the wild-type virus and, most notably, another replication defective mutant that has wild-type levels of polymerase fidelity. These results provide the first experimental evidence that norovirus polymerase fidelity contributes to virus transmission between hosts and that maintaining diversity is important for the establishment of infection. This work supports the hypothesis that the reduced polymerase fidelity of the pandemic GII.4 human norovirus isolates may contribute to their global dominance. IMPORTANCE Virus replication fidelity and hence the intrahost genetic diversity of viral populations are known to be intricately linked to viral pathogenesis and tropism as well as to immune and antiviral escape during infection. In this study, we investigated whether changes in replication fidelity can impact the ability of a virus to transmit between susceptible hosts by the use of a mouse model for norovirus. We show that a variant encoding a high-fidelity polymerase is transmitted less efficiently between mice than the wild-type strain. This constitutes the first experimental demonstration that the polymerase fidelity of viruses can impact transmission of infection in their natural hosts. These results provide further insight into potential reasons for the global emergence of pandemic human noroviruses that display alterations in the replication fidelity of their polymerases compared to nonpandemic strains.

Sign in / Sign up

Export Citation Format

Share Document