scholarly journals Geminivirus betasatellite-encoded βC1 protein exhibits novel ATP hydrolysis activity that influences its DNA-binding activity and viral pathogenesis

2021 ◽  
Author(s):  
Prabu Gnanasekaran ◽  
Neha Gupta ◽  
Kalaiarasan Ponnusamy ◽  
Supriya Chakraborty
Keyword(s):  
Dna Binding ◽  
Atpase Activity ◽  
Viral Pathogenesis ◽  
Binding Activity ◽  
Atp Binding ◽  
Molecular Function ◽  
Dna Binding Activity ◽  
Dominant Negative Mutation ◽  
Arginine Residues ◽  
Pathogenicity Determinant

Plant virus satellites are maintained by their associated helper viruses and satellites influence viral pathogenesis. Diseases caused by geminivirus-betasatellite complexes can become epidemics and therefore have become a threat to economically important crops across the world. Here, we identified a novel molecular function of the betasatellite-encoded pathogenicity determinant βC1. The tomato leaf curl Patna betasatellite (ToLCPaB)-encoded βC1 protein was found to exhibit novel ATPase activity in the presence of the divalent metal ion cofactor MgCl 2 . Moreover, ATPase activity was confirmed to be ubiquitously displayed by βC1 proteins encoded by diverse betasatellites. Mutational and sequence analysis revealed conserved lysine/arginine residues at positions 49/50 and 91 of βC1 proteins to be essential for their ATPase activity. Biochemical studies revealed the DNA-binding activity of the βC1 protein was interfered by the binding of ATP to the protein. Mutating arginine 91 of βC1 to alanine reduced its DNA-binding activity. The results of docking studies provided evidence for an overlap of the ATP-binding and DNA-binding regions of βC1 and for the importance of arginine 91 for both ATP-binding and DNA-binding activities. A mutant betasatellite with a specifically βC1-ATPase dominant negative mutation was found to induce symptoms on Nicotiana benthamiana plants similar to those induced by wild-type betasatellite infection. The ATPase function of βC1 was found to be negatively associated with geminivirus-betasatellite DNA accumulation, despite the positive influence of this ATPase function on the accumulation of replication-associated protein (Rep) and βC1 transcripts. Importance Most satellites influence the pathogenesis of their helper viruses. Here we characterized the novel molecular function of βC1, a non-structural, pathogenicity determinant protein encoded by a betasatellite. Here, we demonstrated the display of ATPase activity by this βC1 protein. Additionally, we confirmed the ubiquitous display of ATPase activity by βC1 proteins encoded by diverse betasatellites. The lysine/arginine residues conserved at positions 49 and 91 of βC1 were found to be crucial for its ATPase function. DNA-binding activity of βC1 was found to be reduced in the presence of ATP. Inhibition of ATPase activity of βC1 in the presence of an excess concentration of cold ATP, GTP, CTP or UTP suggested that the purified βC1 can also hydrolyze other cellular NTPs besides ATP in vitro. These results established the importance of the ATPase and DNA-binding activities of the βC1 protein in regulating the geminivirus-betasatellite DNA accumulation in the infected plant cell.

scholarly journals Role of ATP-binding motifs on DNA-binding activity and biological function of Rhp51, a Rad51 homologue in fission yeast

2002 ◽  
Vol 364 (3) ◽  
pp. 869-874 ◽  
Author(s):  
Woo J. KIM ◽  
Hyojin LEE ◽  
Eon J. PARK ◽  
Seung H. HONG ◽  
Sang D. PARK
Keyword(s):  
Dna Binding ◽  
Binding Activity ◽  
Single Amino Acid ◽  
Atp Binding ◽  
Single Mutation ◽  
Binding Ability ◽  
Binding Motifs ◽  
Dna Binding Activity

Rhp51, a RecA and Rad51 homologue of Schizosaccharomyces pombe, plays a pivotal role in homologous recombination and recombinational repair. It has a set of the well-conserved type A and type B ATP-binding motifs, which are highly conserved in all RecA homologues. In a previous study [Kim, Lee, Park, Park and Park (2001) Nucleic Acids Res. 29, 1724–1732], we reported that a single mutation of the conserved lysine in A motif [Lys155→Ala (K155A)] destroyed the DNA repair ability of Rhp51 and that overexpression of this mutant protein conferred dominant negativity. In the present paper, we investigated DNA-binding properties of recombinant Rhp51 and its mutant proteins. Purified Rhp51 protein showed ATP-dependent double- and single-strand DNA-binding activities. To characterize the role of ATP-binding motifs, we generated Rhp51 K155A and Rhp51 Asp244→Gln (D244Q), which have a single amino acid substitution in A and B motifs respectively. Interestingly, K155A and D244Q mutations impaired ATP-dependent DNA binding in a different manner. K155A lost the DNA binding itself, whereas D244Q maintained the binding ability but lost the ATP dependency. However, despite the difference in DNA-binding ability, both mutations failed to rescue the methylmethane sulphonate and UV sensitivity of the rhp51Δ mutant. Together, these results suggested that not only the DNA binding but also the ATP dependence in DNA binding is required for proper in vivo functioning of Rhp51.

Modulation of Hsf1 activity by novobiocin and geldanamycin

2009 ◽  
Vol 87 (6) ◽  
pp. 845-851 ◽  
Author(s):  
Renaud Conde ◽  
Zachery R. Belak ◽  
Manoj Nair ◽  
Ruth F. O’Carroll ◽  
Nick Ovsenek
Keyword(s):  
Heat Shock ◽  
Dna Binding ◽  
Molecular Mechanisms ◽  
Binding Pocket ◽  
Binding Activity ◽  
Chaperone Activity ◽  
Atp Binding ◽  
Dna Binding Activity ◽  
Culture Cells ◽  
Dose Dependent Decrease

Since Hsp90 is a known modulator of HSF1 activity, we examined the effects of two pharmacological inhibitors of Hsp90, novobiocin and geldanamycin, on HSF1 DNA-binding activity in the Xenopus oocyte model system. Novobiocin exhibits antiproliferative activity in culture cells and interacts with a C-terminal ATP-binding pocket on Hsp90, inhibiting Hsp90 autophosphorylation. Treatment of oocytes with novobiocin followed by heat shock results in a dose-dependent decrease in HSF1 DNA-binding and transcriptional activity. Immunoprecipitation experiments demonstrate novobiocin does not alter HSF1 activity through dissociation of Hsp90 from either monomeric or trimerized HSF1, suggesting that the effect of novobiocin on HSF1 is mediated through alterations in Hsp90 autophosphorylation. Geldanamycin binds the N-terminal ATPase site of Hsp90 and inhibits chaperone activity. Geldanamycin treatment of oocytes resulted in a dose-dependant increase in stability of active HSF1 trimers during submaximal heat shock and a delay in disassembly of trimers during recovery. The results suggest that Hsp90 chaperone activity is required for disassembly of HSF1 trimers. The data obtained with novobiocin suggests the C-terminal ATP-binding activity of Hsp90 is required for the initial steps of HSF1 trimerization, whereas the effects of geldanamycin suggest N-terminal ATPase and chaperone activities are required for disassembly of activated trimers. These data provide important insight into the molecular mechanisms by which pharmacological inhibitors of Hsp90 affect the heat shock response.

Altered expression and insulin-induced trafficking of Na+-K+-ATPase in rat skeletal muscle: effects of high-fat diet and exercise

2009 ◽  
Vol 297 (1) ◽  
pp. E38-E49 ◽  
Author(s):  
Dana Galuska ◽  
Olga Kotova ◽  
Romain Barrès ◽  
Daria Chibalina ◽  
Boubacar Benziane ◽  
...  
Keyword(s):  
Skeletal Muscle ◽  
Transcription Factor ◽  
Dna Binding ◽  
Protein Expression ◽  
Atpase Activity ◽  
Binding Activity ◽  
Insulin Resistant ◽  
Dna Binding Activity ◽  
Skeletal Muscle Insulin Resistance ◽  
Subunit Expression

Skeletal muscle Na+-K+-ATPase plays a central role in the clearance of K+ from the extracellular fluid, therefore maintaining blood [K+]. Na+-K+-ATPase activity in peripheral tissue is impaired in insulin resistant states. We determined effects of high-fat diet (HFD) and exercise training (ET) on skeletal muscle Na+-K+-ATPase subunit expression and insulin-stimulated translocation. Skeletal muscle expression of Na+-K+-ATPase isoforms and transcription factor DNA binding was determined before or after 5 days of swim training in Wistar rats fed chow or HFD for 4 or 12 wk. Skeletal muscle insulin resistance was observed after 12 wk of HFD. Na+-K+-ATPase α1-subunit protein expression was increased 1.6-fold ( P < 0.05), whereas α2- and β1-subunits and protein expression were decreased twofold ( P < 0.01) in parallel with decrease in plasma membrane Na+-K+-ATPase activity after 4 wk of HFD. Exercise training restored α1-, α2-, and β1-subunit expression and Na+-K+-ATPase activity to control levels and reduced β2-subunit expression 2.2-fold ( P < 0.05). DNA binding activity of the α1-subunit-regulating transcription factor ZEB (AREB6) and α1 mRNA expression were increased after HFD and restored by ET. DNA binding activity of Sp-1, a transcription factor involved in the regulation of α2- and β1-subunit expression, was decreased after HFD. ET increased phosphorylation of the Na+-K+-ATPase regulatory protein phospholemman. Phospholemman mRNA and protein expression were increased after HFD and restored to control levels after ET. Insulin-stimulated translocation of the α2-subunit to plasma membrane was impaired by HFD, whereas α1-subunit translocation remained unchanged. Alterations in sodium pump function precede the development of skeletal muscle insulin resistance. Disturbances in skeletal muscle Na+-K+-ATPase regulation, particularly the α2-subunit, may contribute to impaired ion homeostasis in insulin-resistant states such as obesity and type 2 diabetes.

scholarly journals Structure of the p53/RNA polymerase II assembly

2021 ◽  
Vol 4 (1) ◽  
Author(s):  
Shu-Hao Liou ◽  
Sameer K. Singh ◽  
Robert H. Singer ◽  
Robert A. Coleman ◽  
Wei-Li Liu
Keyword(s):  
Dna Binding ◽  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Conformational Changes ◽  
P53 Protein ◽  
Binding Activity ◽  
Transactivation Domain ◽  
Pol Ii ◽  
Dna Binding Activity ◽  
Regulated Gene Expression

AbstractThe tumor suppressor p53 protein activates expression of a vast gene network in response to stress stimuli for cellular integrity. The molecular mechanism underlying how p53 targets RNA polymerase II (Pol II) to regulate transcription remains unclear. To elucidate the p53/Pol II interaction, we have determined a 4.6 Å resolution structure of the human p53/Pol II assembly via single particle cryo-electron microscopy. Our structure reveals that p53’s DNA binding domain targets the upstream DNA binding site within Pol II. This association introduces conformational changes of the Pol II clamp into a further-closed state. A cavity was identified between p53 and Pol II that could possibly host DNA. The transactivation domain of p53 binds the surface of Pol II’s jaw that contacts downstream DNA. These findings suggest that p53’s functional domains directly regulate DNA binding activity of Pol II to mediate transcription, thereby providing insights into p53-regulated gene expression.

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