Distinct Domain Functions Regulating de Novo DNA Synthesis of Thermostable DNA Primase from HyperthermophilePyrococcus horikoshii†

Biochemistry ◽  
2003 ◽  
Vol 42 (50) ◽  
pp. 14968-14976 ◽  
Author(s):  
Eriko Matsui ◽  
Miho Nishio ◽  
Hideshi Yokoyama ◽  
Kazuaki Harata ◽  
Sophie Darnis ◽  
...  
Keyword(s):  
Dna Synthesis ◽  
De Novo ◽  
Dna Primase

scholarly journals An archaeal primase functions as a nanoscale caliper to define primer length

2018 ◽  
Vol 115 (26) ◽  
pp. 6697-6702 ◽  
Author(s):  
Jiangyu Yan ◽  
Sandro Holzer ◽  
Luca Pellegrini ◽  
Stephen D. Bell
Keyword(s):  
Dna Replication ◽  
Dna Synthesis ◽  
De Novo ◽  
Dna Polymerases ◽  
Measuring System ◽  
Structural Homology ◽  
Dna Primase ◽  
Dna And Rna ◽  
Hand Off ◽  
Rna And Dna

The cellular replicative DNA polymerases cannot initiate DNA synthesis without a priming 3′ OH. During DNA replication, this is supplied in the context of a short RNA primer molecule synthesized by DNA primase. The primase of archaea and eukaryotes, despite having varying subunit compositions, share sequence and structural homology. Intriguingly, archaeal primase has been demonstrated to possess the ability to synthesize DNA de novo, a property shared with the eukaryotic PrimPol enzymes. The dual RNA and DNA synthetic capabilities of the archaeal DNA primase have led to the proposal that there may be a sequential hand-off between these synthetic modes of primase. In the current work, we dissect the functional interplay between DNA and RNA synthetic modes of primase. In addition, we determine the key determinants that govern primer length definition by the archaeal primase. Our results indicate a primer measuring system that functions akin to a caliper.

Abstract 396: Regulation of Cardiomyocyte Proliferation by the Hippo Pathway and Dystrophin Complex

2016 ◽  
Vol 119 (suppl_1) ◽  
Author(s):  
Yuka Morikawa ◽  
John Leach ◽  
Todd Heallen ◽  
Ge Tao ◽  
James F Martin
Keyword(s):  
Cell Cycle ◽  
Muscular Dystrophy ◽  
Dna Synthesis ◽  
De Novo ◽  
Hippo Pathway ◽  
Myocardial Damage ◽  
Heart Regeneration ◽  
Cardiomyocyte Proliferation ◽  
Heart Repair ◽  
The Hippo Pathway

Regeneration in mammalian hearts is limited due to the extremely low renewal rate of cardiomyocytes and their inability to reenter the cell cycle. In rodent hearts, endogenous regenerative capacity exists during development but is rapidly repressed after birth, at which time growth is by hypertrophy. During the developmental and neonatal periods, heart regeneration occurs through proliferation of pre-existing cardiomyocytes. Our approach of activating heart regeneration is to uncover the mechanisms responsible for repression of cardiomyocyte proliferation. The Hippo pathway controls heart size by repressing cardiomyocyte proliferation during development. By deleting Salv , a modulator of the Hippo pathway, we found that myocardial damage in postnatal and adult hearts was repaired both anatomically and functionally. This heart repair occurred primary through proliferation of preexisting cardiomyocytes. During repair, cardiomyocytes reenter the cell cycle; de novo DNA synthesis, karyokinesis, and cytokinesis all take place. The dystrophin glycoprotein complex (DGC) is essential for muscle maintenance by anchoring the cytoskeleton and extracellular matrix. Disruption of the DGC results in muscular dystrophies, including Duchenne muscular dystrophy, resulting in both skeletal and cardiac myopathies. Recently the DGC was shown to regulate cardiomyocyte proliferation and we found that the DGC and the Hippo pathway components directly interact. To address if the DGC and the Hippo pathway coordinately regulate cardiomyocyte proliferation, we conditionally deleted Salv in the mouse model of muscular dystrophy, the mdx line. We found that simultaneous disruption of both the DGC and Hippo pathway leads an increased de novo DNA synthesis and cytokinesis in cardiomyocytes after heart damage. Our findings provide new insights into the mechanisms leading to heart repair through proliferation of endogenous cardiomyocytes.

Dexamethasone modulates ErbB tyrosine kinase expression and signaling through multiple and redundant mechanisms in cultured rat hepatocytes

2007 ◽  
Vol 293 (3) ◽  
pp. G552-G559 ◽  
Author(s):  
Lawrence A. Scheving ◽  
Renee Buchanan ◽  
Michael A. Krause ◽  
Xiuqi Zhang ◽  
Mary C. Stevenson ◽  
...  
Keyword(s):  
Growth Factor ◽  
Dna Synthesis ◽  
Transforming Growth Factor ◽  
De Novo ◽  
Rat Hepatocytes ◽  
Immunological Methods ◽  
High Dose ◽  
Transforming Growth Factor Α ◽  
High Dose Dexamethasone ◽  
Cultured Rat Hepatocytes

Glucocorticoids paradoxically exert both stimulatory and inhibitory effects on the proliferation of cultured rat hepatocytes. We studied the effects of dexamethasone, a synthetic glucocorticoid, on the proliferation of cultured rat hepatocytes. The timing of growth factor addition modified the action of high-dose dexamethasone (10−6 M) on DNA synthesis. When we added transforming growth factor-α at the time of plating, 10−6 M dexamethasone weakly stimulated DNA synthesis by 26% relative to cells cultured in dexamethasone-free media. When we delayed growth factor addition until 24–48 h after plating, 10−6 M dexamethasone inhibited DNA synthesis by 50%. Using immunological methods, we analyzed the expression and signaling patterns of the ErbB kinases in dexamethasone-treated cells. High-dose dexamethasone stabilized the expression of epidermal growth factor receptor (EGFr) and ErbB3, and it suppressed the de novo expression of ErbB2 that occurs during the third and fourth day of culture in 10−8 M dexamethasone. High-dose dexamethasone by 72 h suppressed basal and EGF-associated phosphorylation of ERK and Akt. The reduction in ERK1/2 phosphorylation correlated with suppression of a culture-dependent increase in Son-of sevenless 1 (Sos1) and ERK1/2 expression. High-dose dexamethasone in hepatocytes stabilized or upregulated several inhibitory effectors of EGFr/ErbB2 and ERK, including receptor-associated late transducer (RALT) and MKP-1, respectively. Thus 10−6 M dexamethasone exerts a time-dependent and redundant inhibitory effect on EGFr-mediated proliferative signaling in hepatocytes, targeting not only the ErbB proteins but also their various positive and negative effectors.

De Novo DNA Synthesis Using Single-Molecule PCR

2012 ◽  
pp. 35-47 ◽  
Author(s):  
Tuval Ben Yehezkel ◽  
Gregory Linshiz ◽  
Ehud Shapiro
Keyword(s):  
Dna Synthesis ◽  
Single Molecule ◽  
De Novo

Stretch-mediated visceral smooth muscle growth in vitro

1992 ◽  
Vol 262 (5) ◽  
pp. R895-R900
Author(s):  
O. M. Karim ◽  
K. Pienta ◽  
N. Seki ◽  
J. L. Mostwin
Keyword(s):  
Smooth Muscle ◽  
Dna Synthesis ◽  
De Novo ◽  
Specific Activity ◽  
Muscle Growth ◽  
Mechanical Stimulus ◽  
Trophic Factors ◽  
Taenia Coli ◽  
Visceral Smooth Muscle

An in vitro model of smooth muscle stretch was developed to study mechanical stimulus as a possible mediator of visceral smooth muscle growth and differences in the growth response of smooth muscle from young and old animals. De novo DNA synthesis as measured by the aphidicolin-sensitive specific activity of DNA was used as an index of cell growth. Compared with old tissue, the rate of aphidicolin-sensitive DNA synthesis in smooth muscle from young animals was 3-5 and 1.5-2 times greater in bladder and taenia coli, respectively. Stretch of bladder muscle and taenia coli strips from young animals for 6 h increased the aphidicolin-sensitive specific activity of DNA 3-fold (P less than 0.01) and 1.5-fold (P less than 0.01), respectively. Tissue from old animals, however, under the same conditions increased the rate of aphidicolin-resistant DNA synthesis, possibly implying DNA repair. Autoradiography showed only labeled myocyte nuclei. These results indicate that homeostatic mechanisms modulating myocyte growth in visceral smooth muscle can respond to mechanical stimulus in the absence of other trophic factors.

Apoptosis in megaloblastic anemia occurs during DNA synthesis by a p53-independent, nucleoside-reversible mechanism

Blood ◽  
2000 ◽  
Vol 96 (9) ◽  
pp. 3249-3255 ◽  
Author(s):  
Mark J. Koury ◽  
James O. Price ◽  
Geoffrey G. Hicks
Keyword(s):  
Dna Synthesis ◽  
De Novo ◽  
Megaloblastic Anemia ◽  
Experimental Studies ◽  
Hematopoietic Cells ◽  
S Phase ◽  
De Novo Synthesis ◽  
Complete Reversal ◽  
Phase Accumulation

Abstract Deficiency of folate or vitamin B12 (cobalamin) causes megaloblastic anemia, a disease characterized by pancytopenia due to the excessive apoptosis of hematopoietic progenitor cells. Clinical and experimental studies of megaloblastic anemia have demonstrated an impairment of DNA synthesis and repair in hematopoietic cells that is manifested by an increased percentage of cells in the DNA synthesis phase (S phase) of the cell cycle, compared with normal hematopoietic cells. Both folate and cobalamin are required for normal de novo synthesis of thymidylate and purines. However, previous studies of impaired DNA synthesis and repair in megaloblastic anemia have concerned mainly the decreased intracellular levels of thymidylate and its effects on nucleotide pools and misincorporation of uracil into DNA. An in vitro model of folate-deficient erythropoiesis was used to study the relationship between the S-phase accumulation and apoptosis in megaloblastic anemia. The results indicate that folate-deficient erythroblasts accumulate in and undergo apoptosis in the S phase when compared with control erythroblasts. Both the S-phase accumulation and the apoptosis were induced by folate deficiency in erythroblasts fromp53 null mice. The complete reversal of the S-phase accumulation and apoptosis in folate-deficient erythroblasts required the exogenous provision of specific purines or purine nucleosides as well as thymidine. These results indicate that decreased de novo synthesis of purines plays as important a role as decreased de novo synthesis of thymidylate in the pathogenesis of megaloblastic anemia.

scholarly journals MESA: automated assessment of synthetic DNA fragments and simulation of DNA synthesis, storage, sequencing and PCR errors

2020 ◽  
Vol 36 (11) ◽  
pp. 3322-3326
Author(s):  
Michael Schwarz ◽  
Marius Welzel ◽  
Tolganay Kabdullayeva ◽  
Anke Becker ◽  
Bernd Freisleben ◽  
...  
Keyword(s):  
Dna Synthesis ◽  
Web Application ◽  
De Novo ◽  
Supplementary Information ◽  
Dna Fragments ◽  
Synthetic Dna ◽  
Custom Made ◽  
Dna Fragment ◽  
Polymerase Chain ◽  
Error Probabilities

Abstract Summary The development of de novo DNA synthesis, polymerase chain reaction (PCR), DNA sequencing and molecular cloning gave researchers unprecedented control over DNA and DNA-mediated processes. To reduce the error probabilities of these techniques, DNA composition has to adhere to method-dependent restrictions. To comply with such restrictions, a synthetic DNA fragment is often adjusted manually or by using custom-made scripts. In this article, we present MESA (Mosla Error Simulator), a web application for the assessment of DNA fragments based on limitations of DNA synthesis, amplification, cloning, sequencing methods and biological restrictions of host organisms. Furthermore, MESA can be used to simulate errors during synthesis, PCR, storage and sequencing processes. Availability and implementation MESA is available at mesa.mosla.de, with the source code available at github.com/umr-ds/mesa_dna_sim. Contact [email protected] Supplementary information Supplementary data are available at Bioinformatics online.

Distinct PKC isoforms mediate cell survival and DNA synthesis in thrombin-induced myofibroblasts

2005 ◽  
Vol 288 (1) ◽  
pp. L190-L201 ◽  
Author(s):  
Galina S. Bogatkevich ◽  
Estella Gustilo ◽  
Jim C. Oates ◽  
Carol Feghali-Bostwick ◽  
Russell A. Harley ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Dna Synthesis ◽  
Cyclin D1 ◽  
Fas Ligand ◽  
De Novo ◽  
Regulatory Protein ◽  
Mitogen Activated Protein Kinase ◽  
Lung Fibroblasts ◽  
Normal Lung ◽  
Pkc Isoforms

Thrombin activates protease-activated receptor (PAR)-1 and induces a myofibroblast phenotype in normal lung fibroblasts that resembles the phenotype of scleroderma lung fibroblasts. We now demonstrate that PAR-1 expression is dramatically increased in lung tissue from scleroderma patients, where it is associated with inflammatory and fibroproliferative foci. We also observe that thrombin induces resistance to apoptosis in normal lung fibroblasts, and this process is regulated by protein kinase C (PKC)-ε but not by PKC-α. Overexpression of a constitutively active (c-a) form of PAR-1 or PKC-ε significantly inhibits Fas ligand-induced apoptosis in lung fibroblasts, whereas scleroderma lung fibroblasts are resistant to apoptosis de novo. Thrombin translocates p21Cip1/WAF1, a signaling molecule downstream of PKC, from the nucleus to cytoplasm in normal lung fibroblasts mimicking the localization of p21Cip1/WAF1 in scleroderma lung fibroblasts. Overexpression of c-a PKC-α or PKC-ε results in accumulation of p21Cip1/WAF1 in the cytoplasm. Depletion of PKC-α or inhibition of mitogen-activated protein kinase (MAPK) blocks thrombin-induced DNA synthesis in lung fibroblasts. Inhibition of PKC by calphostin or PKC-α, but not PKC-ε, by antisense oligonucleotides prevents thrombin-induced MAPK phosphorylation and accumulation of G1 phase regulatory protein cyclin D1, suggesting that PKC-α, MAPK, and cyclin D1 mediate lung fibroblast proliferation. These data demonstrate that two distinct PKC isoforms mediate thrombin-induced resistance to apoptosis and proliferation and suggest that p21Cip1/WAF1 promotes both phenomena.

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