scholarly journals The large terminase DNA packaging motor grips DNA with its ATPase domain for cleavage by the flexible nuclease domain

2016 ◽  
Author(s):  
Brendan J. Hilbert ◽  
Janelle A. Hayes ◽  
Nicholas P. Stone ◽  
Rui-Gang Xu ◽  
Brian A. Kelch
Keyword(s):  
Dna Binding ◽  
Active Site ◽  
Dna Cleavage ◽  
Atpase Domain ◽  
Dna Translocation ◽  
Genome Packaging ◽  
Virus Maturation ◽  
Nuclease Domain ◽  
Dna Packaging Motor ◽  
Large Terminase

AbstractMany viruses use a powerful terminase motor to pump their genome inside an empty procapsid shell during virus maturation. The large terminase (TerL) protein contains both enzymatic activities necessary for packaging in such viruses: the ATPase that powers DNA translocation and an endonuclease that cleaves the concatemeric genome both at initiation and completion of genome packaging. However, how TerL binds DNA during translocation and cleavage is still mysterious. Here we investigate DNA binding and cleavage using TerL from the thermophilic phage P74-26. We report the structure of the P74-26 TerL nuclease domain, which allows us to model DNA binding in the nuclease active site. We screened a large panel of TerL variants for defects in binding and DNA cleavage, revealing that the ATPase domain is the primary site for DNA binding, and is required for nucleolysis. The nuclease domain is dispensable for DNA binding but residues lining the active site guide DNA for cleavage. Kinetic analysis of nucleolysis suggests flexible tethering of the nuclease domains during DNA cleavage. We propose that interactions with the procapsid shell during DNA translocation conformationally restrict the nuclease domain, inhibiting cleavage; TerL release from the procapsid upon completion of packaging unlocks the nuclease domains to cleave DNA.

scholarly journals The large terminase DNA packaging motor grips DNA with its ATPase domain for cleavage by the flexible nuclease domain

2017 ◽  
pp. gkw1356 ◽  
Author(s):  
Brendan J. Hilbert ◽  
Janelle A. Hayes ◽  
Nicholas P. Stone ◽  
Rui-Gang Xu ◽  
Brian A. Kelch
Keyword(s):  
Dna Packaging ◽  
Atpase Domain ◽  
Nuclease Domain ◽  
Dna Packaging Motor ◽  
Large Terminase

scholarly journals The interplay of DNA binding, ATP hydrolysis and helicase activities of the archaeal MCM helicase

2011 ◽  
Vol 436 (2) ◽  
pp. 409-414 ◽  
Author(s):  
Li Phing Liew ◽  
Stephen D. Bell
Keyword(s):  
Dna Binding ◽  
Active Site ◽  
Atp Hydrolysis ◽  
Sulfolobus Solfataricus ◽  
Active Form ◽  
Dna Helicase ◽  
Atpase Domain ◽  
Minichromosome Maintenance ◽  
Conserved Residues ◽  
Mcm Helicase

The MCM (minichromosome maintenance) proteins of archaea are widely believed to be the replicative DNA helicase of these organisms. Most archaea possess a single MCM orthologue that forms homo-multimeric assemblies with a single hexamer believed to be the active form. In the present study we characterize the roles of highly conserved residues in the ATPase domain of the MCM of the hyperthermophilic archaeon Sulfolobus solfataricus. Our results identify a potential conduit for communicating DNA-binding information to the ATPase active site.

scholarly journals A viral genome packaging ring-ATPase is a flexibly coordinated pentamer

2021 ◽  
Author(s):  
Li Dai ◽  
Digvijay Singh ◽  
Suoang Lu ◽  
Vishal Kottadiel ◽  
Reza Vafabakhsh ◽  
...  
Keyword(s):  
Single Molecule ◽  
Viral Genome ◽  
Motor Coordination ◽  
Bacteriophage T4 ◽  
Dna Packaging ◽  
Biological Functions ◽  
Dna Translocation ◽  
Genome Packaging ◽  
Dna Packaging Motor ◽  
Direct Counting

Multi-subunit ring-ATPases carry out a myriad of biological functions, including genome packaging in viruses. Though the basic structures and functions of these motors have been well-established, the mechanisms of ATPase firing and motor coordination are poorly understood. Here, by direct counting using single-molecule fluorescence, we have determined that the active bacteriophage T4 DNA packaging motor consists of five subunits of gp17. By systematically doping motors with an ATPase-defective subunit and selecting single motors containing a precise count of active/inactive subunit(s), we found, unexpectedly, that the packaging motor can tolerate an inactive sub-unit. However, motors containing an inactive subunit(s) exhibit fewer DNA engagements, a higher failure rate in encapsidation, reduced packaging velocity, and increased pausing. These findings suggest a new packaging model in which the motor, by re-adjusting its grip on DNA, can skip an inactive subunit and resume DNA translocation, contrary to the prevailing notion of strict coordination amongst motor subunits of other packaging motors.

scholarly journals A viral genome packaging ring-ATPase is a flexibly coordinated pentamer

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Li Dai ◽  
Digvijay Singh ◽  
Suoang Lu ◽  
Vishal I. Kottadiel ◽  
Reza Vafabakhsh ◽  
...  
Keyword(s):  
Single Molecule ◽  
Viral Genome ◽  
Motor Coordination ◽  
Bacteriophage T4 ◽  
Dna Packaging ◽  
Biological Functions ◽  
Dna Translocation ◽  
Single Molecule Fluorescence ◽  
Genome Packaging ◽  
Dna Packaging Motor

AbstractMulti-subunit ring-ATPases carry out a myriad of biological functions, including genome packaging in viruses. Though the basic structures and functions of these motors have been well-established, the mechanisms of ATPase firing and motor coordination are poorly understood. Here, using single-molecule fluorescence, we determine that the active bacteriophage T4 DNA packaging motor consists of five subunits of gp17. By systematically doping motors with an ATPase-defective subunit and selecting single motors containing a precise number of active or inactive subunits, we find that the packaging motor can tolerate an inactive subunit. However, motors containing one or more inactive subunits exhibit fewer DNA engagements, a higher failure rate in encapsidation, reduced packaging velocity, and increased pausing. These findings suggest a DNA packaging model in which the motor, by re-adjusting its grip on DNA, can skip an inactive subunit and resume DNA translocation, suggesting that strict coordination amongst motor subunits of packaging motors is not crucial for function.

scholarly journals Structure-Function Analysis of IntDOT

2009 ◽  
Vol 192 (2) ◽  
pp. 575-586 ◽  
Author(s):  
Seyeun Kim ◽  
Brian M. Swalla ◽  
Jeffrey F. Gardner
Keyword(s):  
Homology Modeling ◽  
Protein Interactions ◽  
Active Site ◽  
Dna Cleavage ◽  
Function Analysis ◽  
Indicator Strain ◽  
Amino Terminal ◽  
Dna Ligation ◽  
Polypeptide Backbone

ABSTRACT CTnDOT integrase (IntDOT) is a member of the tyrosine family of site-specific DNA recombinases. IntDOT is unusual in that it catalyzes recombination between nonidentical sequences. Previous mutational analyses centered on mutants with substitutions of conserved residues in the catalytic (CAT) domain or residues predicted by homology modeling to be close to DNA in the core-binding (CB) domain. That work suggested that a conserved active-site residue (Arg I) of the CAT domain is missing and that some residues in the CB domain are involved in catalysis. Here we used a genetic approach and constructed an Escherichia coli indicator strain to screen for random mutations in IntDOT that disrupt integrative recombination in vivo. Twenty-five IntDOT mutants were isolated and characterized for DNA binding, DNA cleavage, and DNA ligation activities. We found that mutants with substitutions in the amino-terminal (N) domain were catalytically active but defective in forming nucleoprotein complexes, suggesting that they have altered protein-protein interactions or altered interactions with DNA. Replacement of Ala-352 of the CAT domain disrupted DNA cleavage but not DNA ligation, suggesting that Ala-352 may be important for positioning the catalytic tyrosine (Tyr-381) during cleavage. Interestingly, our biochemical data and homology modeling of the CAT domain suggest that Arg-285 is the missing Arg I residue of IntDOT. The predicted position of Arg-285 shows it entering the active site from a position on the polypeptide backbone that is not utilized in other tyrosine recombinases. IntDOT may therefore employ a novel active-site architecture to catalyze recombination.

scholarly journals Synthesis, Characterization, DNA Binding, and Photocleavage Activity of Oxorhenium (V) Complexes withα-Diimine and Quinoxaline Ligands

2010 ◽  
Vol 2010 ◽  
pp. 1-9 ◽  
Author(s):  
Christiana A. Mitsopoulou ◽  
Constantinos Dagas
Keyword(s):  
Cyclic Voltammetry ◽  
Dna Binding ◽  
Dna Cleavage ◽  
2D Nmr ◽  
Base Pairs ◽  
Binding Properties ◽  
Supercoiled Form ◽  
Dna Base ◽  
Dna Base Pairs ◽  
Dna Binding Properties

The complex [ReOCl3pq] (1) (where pq = 2-(2′pyridyl)quinoxaline) has been synthesized and fully characterized by UV-Vis, FTIR, 1 and 2D NMR, and cyclic voltammetry (CV). The DNA-binding properties of the complex1as well as of the compounds [ReOCl3bpy] (2), [ReOCl3phen] (3), and pq (4) were investigated by UV-spectrophotometric (melting curves), CV (cyclic voltammetry), and viscosity measurements. Experimental data suggest that complex1intercalates into the DNA base pairs. Upon irradiation, complex1was found to promote the cleavage of plasmid pBR 322 DNA from supercoiled form I to nicked form II. The mechanism of the DNA cleavage by complex1was also investigated.

scholarly journals Analysis of the HindIII-catalyzed reaction by time-resolved crystallography

2015 ◽  
Vol 71 (2) ◽  
pp. 256-265 ◽  
Author(s):  
Takashi Kawamura ◽  
Tomoki Kobayashi ◽  
Nobuhisa Watanabe
Keyword(s):  
Electron Density ◽  
Active Site ◽  
Dna Cleavage ◽  
Metal Ion ◽  
Manganese Ions ◽  
Metal Ion Binding ◽  
Time Resolved ◽  
Manganese Ion ◽  
Ion Binding Sites ◽  
Metal Ion Binding Sites

In order to investigate the mechanism of the reaction catalyzed by HindIII, structures of HindIII–DNA complexes with varying durations of soaking time in cryoprotectant buffer containing manganese ions were determined by the freeze-trap method. In the crystal structures of the complexes obtained after soaking for a longer duration, two manganese ions, indicated by relatively higher electron density, are clearly observed at the two metal ion-binding sites in the active site of HindIII. The increase in the electron density of the two metal-ion peaks followed distinct pathways with increasing soaking times, suggesting variation in the binding rate constant for the two metal sites. DNA cleavage is observed when the second manganese ion appears, suggesting that HindIII uses the two-metal-ion mechanism, or alternatively that its reactivity is enhanced by the binding of the second metal ion. In addition, conformational change in a loop near the active site accompanies the catalytic reaction.

Syntheses, crystal structures, DNA binding, DNA cleavage and DFT study of Co(iii) complexes involving azo-appended Schiff base ligands

2018 ◽  
Vol 42 (20) ◽  
pp. 16571-16582 ◽  
Author(s):  
Saikat Banerjee ◽  
Roumi Patra ◽  
Pravat Ghorai ◽  
Paula Brandão ◽  
Sougata Ghosh Chowdhury ◽  
...  
Keyword(s):  
Schiff Base ◽  
Dna Binding ◽  
Crystal Structures ◽  
Dna Cleavage ◽  
Dft Study ◽  
Schiff Base Ligands

Herein, we have reported three new Co(iii) complexes involving azo-appended Schiff base ligands.

Sign in / Sign up

Export Citation Format

Share Document