scholarly journals Amidst multiple binding orientations on fork DNA, Saccharolobus MCM helicase proceeds N-first for unwinding

eLife ◽  
2019 ◽  
Vol 8 ◽  
Author(s):  
Himasha M Perera ◽  
Michael A Trakselis
Keyword(s):  
Site Specific ◽  
Winged Helix ◽  
Dna Footprinting ◽  
Single Turnover ◽  
Mcm Helicase ◽  
Multiple Binding ◽  
Dna Strands ◽  
Binding Orientations ◽  
Hexameric Helicases ◽  
Dna Substrate

DNA replication requires that the duplex genomic DNA strands be separated; a function that is implemented by ring-shaped hexameric helicases in all Domains. Helicases are composed of two domains, an N- terminal DNA binding domain (NTD) and a C- terminal motor domain (CTD). Replication is controlled by loading of helicases at origins of replication, activation to preferentially encircle one strand, and then translocation to begin separation of the two strands. Using a combination of site-specific DNA footprinting, single-turnover unwinding assays, and unique fluorescence translocation monitoring, we have been able to quantify the binding distribution and the translocation orientation of Saccharolobus (formally Sulfolobus) solfataricus MCM on DNA. Our results show that both the DNA substrate and the C-terminal winged-helix (WH) domain influence the orientation but that translocation on DNA proceeds N-first.

scholarly journals Staring at the Naked Goddess: Unraveling the Structure and Reactivity of Artemis Endonuclease Interacting with a DNA Double Strand

Molecules ◽  
2021 ◽  
Vol 26 (13) ◽  
pp. 3986
Author(s):  
Cécilia Hognon ◽  
Antonio Monari
Keyword(s):  
Dna Cleavage ◽  
Catalytic Mechanism ◽  
Dna Lesions ◽  
Cleavage Reaction ◽  
Dynamic Simulations ◽  
Important Target ◽  
System Adaptation ◽  
Dna Strands ◽  
Dna Strand ◽  
Dna Substrate

Artemis is an endonuclease responsible for breaking hairpin DNA strands during immune system adaptation and maturation as well as the processing of potentially toxic DNA lesions. Thus, Artemis may be an important target in the development of anticancer therapy, both for the sensitization of radiotherapy and for immunotherapy. Despite its importance, its structure has been resolved only recently, and important questions concerning the arrangement of its active center, the interaction with the DNA substrate, and the catalytic mechanism remain unanswered. In this contribution, by performing extensive molecular dynamic simulations, both classically and at the hybrid quantum mechanics/molecular mechanics level, we evidenced the stable interaction modes of Artemis with a model DNA strand. We also analyzed the catalytic cycle providing the free energy profile and key transition states for the DNA cleavage reaction.

scholarly journals Two-tiered enforcement of high-fidelity DNA ligation

2019 ◽  
Vol 10 (1) ◽  
Author(s):  
Percy P. Tumbale ◽  
Thomas J. Jurkiw ◽  
Matthew J. Schellenberg ◽  
Amanda A. Riccio ◽  
Patrick J O’Brien ◽  
...  
Keyword(s):  
Dna Damage ◽  
Oxidative Dna Damage ◽  
High Fidelity ◽  
Dna Ligases ◽  
Dna Junctions ◽  
Dna Base ◽  
Dna Strands ◽  
Dna Mismatches ◽  
Second Tier ◽  
Dna Substrate

AbstractDNA ligases catalyze the joining of DNA strands to complete DNA replication, recombination and repair transactions. To protect the integrity of the genome, DNA ligase 1 (LIG1) discriminates against DNA junctions harboring mutagenic 3′-DNA mismatches or oxidative DNA damage, but how such high-fidelity ligation is enforced is unknown. Here, X-ray structures and kinetic analyses of LIG1 complexes with undamaged and oxidatively damaged DNA unveil that LIG1 employs Mg2+-reinforced DNA binding to validate DNA base pairing during the adenylyl transfer and nick-sealing ligation reaction steps. Our results support a model whereby LIG1 fidelity is governed by a high-fidelity (HiFi) interface between LIG1, Mg2+, and the DNA substrate that tunes the enzyme to release pro-mutagenic DNA nicks. In a second tier of protection, LIG1 activity is surveilled by Aprataxin (APTX), which suppresses mutagenic and abortive ligation at sites of oxidative DNA damage.

Site-specific Recombination by Tn3Resolvase, Photocrosslinked to Its Supercoiled DNA Substrate

1996 ◽  
Vol 260 (3) ◽  
pp. 299-303 ◽  
Author(s):  
Michael J. McIlwraith ◽  
Martin R. Boocock ◽  
Marshall W. Stark
Keyword(s):  
Supercoiled Dna ◽  
Site Specific ◽  
Site Specific Recombination ◽  
Dna Substrate

scholarly journals Multiple steroid-binding orientations: alteration of regiospecificity of dehydroepiandrosterone 2- and 7-hydroxylase activities of cytochrome P-450 2a-5 by mutation of residue 209

1995 ◽  
Vol 306 (1) ◽  
pp. 29-33 ◽  
Author(s):  
M Iwasaki ◽  
D G Davis ◽  
T A Darden ◽  
L G Pedersen ◽  
M Negishi
Keyword(s):  
Dynamic Equilibrium ◽  
The Other ◽  
Axial Position ◽  
Hydroxylase Activity ◽  
Steroid Molecule ◽  
Multiple Binding ◽  
Critical Residues ◽  
Steroid Binding ◽  
Binding Orientations ◽  
Cytochrome P 450

The mutation of Ala-117 to Val conferred dehydroepiandrosterone (DHEA) hydroxylase activity on cytochrome P-450 2a-4, with the production of both 2 alpha- and 7 alpha-hydroxyDHEA at similar rates. P-450 2a-5 which has Val at position 117, acquired high DHEA hydroxylase activity by mutation of Phe-209. Mutant F209L of P-450 2a-5 exhibited strong regiospecificity at the 2-position of the DHEA molecule with the production of 2 alpha-hydroxy DHEA as the major metabolite. On the other hand, mutant F209V of P-450 2a-5 showed the 7-position to be the major hydroxylation site, 7 beta-hydroxyDHEA and 7 alpha-OHDHEA being produced. Therefore the regiospecificity of DHEA hydroxylase activity of P-450 2a-5 is altered between the 2- and 7-position depending on the amino acid at position 209. Modelling of the DHEA molecule in the pocket of bacterial P-450cam showed that the steroid can be accommodated in at least two orientations for which the 2- or 7- position is near the sixth axial position of the haem. Moreover, these two orientations, which are of similar energy, can be interconverted by a 180 degrees rotation of the steroid molecule around its long axis. These results support the hypothesis that the steroid molecule in the pocket is in dynamic equilibrium with multiple binding orientations and that the equilibrium is apparently determined by a few critical residues including those at positions 117 and 209.

scholarly journals In Vivo Evidence for Two Active Nuclease Motifs in the Double-Strand Break Repair Enzyme RexAB ofLactococcus lactis

2001 ◽  
Vol 183 (13) ◽  
pp. 4071-4078 ◽  
Author(s):  
Andréa Quiberoni ◽  
Indranil Biswas ◽  
Meriem El Karoui ◽  
Lahcen Rezaı̈ki ◽  
Patrick Tailliez ◽  
...  
Keyword(s):  
Nuclease Activity ◽  
Strand Break ◽  
Dsb Repair ◽  
Repair Enzyme ◽  
Site Specific ◽  
Gram Positive Bacteria ◽  
Dna Strands ◽  
Dna Strand ◽  
Regulatory Dna

ABSTRACT In bacteria, double-strand DNA break (DSB) repair involves an exonuclease/helicase (exo/hel) and a short regulatory DNA sequence (Chi) that attenuates exonuclease activity and stimulates DNA repair. Despite their key role in cell survival, these DSB repair components show surprisingly little conservation. The best-studied exo/hel, RecBCD of Escherichia coli, is composed of three subunits. In contrast, RexAB of Lactococcus lactis and exo/hel enzymes of other low-guanine-plus-cytosine branch gram-positive bacteria contain two subunits. We report that RexAB functions via a novel mechanism compared to that of the RecBCD model. Two potential nuclease motifs are present in RexAB compared with a single nuclease in RecBCD. Site-specific mutagenesis of the RexA nuclease motif abolished all nuclease activity. In contrast, the RexB nuclease motif mutants displayed strongly reduced nuclease activity but maintained Chi recognition and had a Chi-stimulated hyperrecombination phenotype. The distinct phenotypes resulting from RexA or RexB nuclease inactivation lead us to suggest that each of the identified active nuclease sites in RexAB is involved in the degradation of one DNA strand. In RecBCD, the single RecB nuclease degrades both DNA strands and is presumably positioned by RecD. The presence of two nucleases would suggest that this RecD function is dispensable in RexAB.

The MCM complex: (just) a replicative helicase?

2008 ◽  
Vol 36 (1) ◽  
pp. 136-140 ◽  
Author(s):  
Alessandro Costa ◽  
Silvia Onesti
Keyword(s):  
Minichromosome Maintenance ◽  
Replicative Helicase ◽  
Aaa Atpase ◽  
Mcm Helicase ◽  
Eukaryotic Dna ◽  
Mcm Complex ◽  
Elongation Step ◽  
Hexameric Helicases

The MCM2–MCM7 (minichromosome maintenance 2–7) complex is involved both in the initiation and the elongation step of eukaryotic DNA replication and is believed to be the replicative helicase. Whereas the mechanism of DNA unwinding at the replication fork has been extensively investigated, the role of the MCM2–MCM7 complex during initiation has not yet been characterized by biochemical studies. Here we summarize the in vivo evidence which supports a role for the MCM complex in origin melting. In addition, we present an overview of the mechanism of action of a number of AAA+ (ATPase associated with various cellular activities) initiators and hexameric helicases, which can be used in turn as models for the steps of recognition, duplex melting, loading and nucleic acid translocation of the MCM helicase.

Modeling of Supramolecular Systems, Mechanically Docked to Carbon Nanotubes

2006 ◽  
Vol 924 ◽  
Author(s):  
Jordan Poler ◽  
T. D. DuBois
Keyword(s):  
Carbon Nanotubes ◽  
Binding Sites ◽  
Three Dimensional ◽  
Optoelectronic Devices ◽  
Two Dimensional ◽  
Supramolecular Systems ◽  
Site Specific ◽  
Multiple Binding Sites ◽  
Multiple Binding ◽  
Multidimensional Constraints

ABSTRACTCarbon nanotubes and nanowires are important materials for new nanotechnology devices and sensors. Future optoelectronic devices can be made from assemblies of nanostructured materials. One difficulty in preparing these assemblies from nanotubes is the lack of site-specific points of contact and the subsequent compliance of the linkage between nanoparticles. Using molecular mechanics, semiempirical and dynamics calculations, we have modeled the assembly process of two-dimensional and three-dimensional structures of carbon nanotubes. The linkers between the nanotubes consist of novel metallodendrimers. These dendrimers have multiple binding sites with chemically specified chirality. Most importantly, they are mechanically rigid. This enables the multidimensional constraints and geometry, required for advanced electronic and optoelectronic devices.

scholarly journals Binding Orientations and Lipid Interactions of Human Amylin at Zwitterionic and Anionic Lipid Bilayers

2016 ◽  
Vol 2016 ◽  
pp. 1-13 ◽  
Author(s):  
Zhenyu Qian ◽  
Yan Jia ◽  
Guanghong Wei
Keyword(s):  
Lipid Bilayers ◽  
Helical Structure ◽  
Terminal Region ◽  
Islet Amyloid ◽  
Anionic Lipid ◽  
Multiple Binding ◽  
Preferential Binding ◽  
Dynamics Simulations ◽  
Binding Orientations ◽  
Human Amylin

Increasing evidence suggests that the interaction of human islet amyloid polypeptide (hIAPP) with lipids may facilitate hIAPP aggregation and cause the death of pancreatic isletβ-cells. However, the detailed hIAPP-membrane interactions and the influences of lipid compositions are unclear. In this study, as a first step to understand the mechanism of membrane-mediated hIAPP aggregation, we investigate the binding behaviors of hIAPP monomer at zwitterionic palmitoyloleoyl-phosphatidylcholine (POPC) bilayer by performing atomistic molecular dynamics simulations. The results are compared with those of hIAPP at anionic palmitoyloleoyl-phosphatidylglycerol (POPG) bilayers. We find that the adsorption of hIAPP to POPC bilayer is mainly initiated from the C-terminal region and the peptide adopts a helical structure with multiple binding orientations, while the adsorption to POPG bilayer is mostly initiated from the N-terminal region and hIAPP displays one preferential binding orientation, with its hydrophobic residues exposed to water. hIAPP monomer inserts into POPC lipid bilayers more readily than into POPG bilayers. Peptide-lipid interaction analyses show that the different binding features of hIAPP at POPC and POPG bilayers are attributed to different magnitudes of electrostatic and hydrogen-bonding interactions with lipids. This study provides mechanistic insights into the different interaction behaviors of hIAPP with zwitterionic and anionic lipid bilayers.

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