scholarly journals Structural Polymorphism of Single pDNA Condensates Elicited by Cationic Block Polyelectrolytes

Polymers ◽  
2020 ◽  
Vol 12 (7) ◽  
pp. 1603
Author(s):  
Kensuke Osada
Keyword(s):  
Single Molecule ◽  
Structural Polymorphism ◽  
Genome Packaging ◽  
Dna Folding ◽  
Polyelectrolyte Complexation ◽  
Gene Vectors ◽  
Cationic Copolymers ◽  
Complex Forms ◽  
Dna Complex ◽  
Systemic Application

DNA folding is a core phenomenon in genome packaging within a nucleus. Such a phenomenon is induced by polyelectrolyte complexation between anionic DNA and cationic proteins of histones. In this regard, complexes formed between DNA and cationic polyelectrolytes have been investigated as models to gain insight into genome packaging. Upon complexation, DNA undergoes folding to reduce its occupied volume, which often results in multi-complex associated aggregates. However, when cationic copolymers comprising a polycation block and a neutral hydrophilic polymer block are used instead, DNA undergoes folding as a single molecule within a spontaneously formed polyplex micelle (PM), thereby allowing the observation of the higher-order structures that DNA forms. The DNA complex forms polymorphic structures, including globular, rod-shaped, and ring-shaped (toroidal) structures. This review focuses on the polymorphism of DNA, particularly, to elucidate when, how, and why DNA organizes into these structures with cationic copolymers. The interactions between DNA and the copolymers, and the specific nature of DNA in rigidity; i.e., rigid but foldable, play significant roles in the observed polymorphism. Moreover, PMs serve as potential gene vectors for systemic application. The significance of the controlled DNA folding for such an application is addressed briefly in the last part.

scholarly journals Using single-molecule FRET to probe the nucleotide-dependent conformational landscape of polymerase β-DNA complexes

2020 ◽  
Vol 295 (27) ◽  
pp. 9012-9020
Author(s):  
Carel Fijen ◽  
Mariam M. Mahmoud ◽  
Meike Kronenberg ◽  
Rebecca Kaup ◽  
Mattia Fontana ◽  
...  
Keyword(s):  
Dna Repair ◽  
Single Molecule ◽  
Conformational Changes ◽  
Dna Complexes ◽  
Single Molecule Fret ◽  
Nucleotide Incorporation ◽  
Eukaryotic Dna ◽  
Cellular Dna ◽  
Dna Complex ◽  
Gapped Dna

Eukaryotic DNA polymerase β (Pol β) plays an important role in cellular DNA repair, as it fills short gaps in dsDNA that result from removal of damaged bases. Since defects in DNA repair may lead to cancer and genetic instabilities, Pol β has been extensively studied, especially its mechanisms for substrate binding and a fidelity-related conformational change referred to as “fingers closing.” Here, we applied single-molecule FRET to measure distance changes associated with DNA binding and prechemistry fingers movement of human Pol β. First, using a doubly labeled DNA construct, we show that Pol β bends the gapped DNA substrate less than indicated by previously reported crystal structures. Second, using acceptor-labeled Pol β and donor-labeled DNA, we visualized dynamic fingers closing in single Pol β-DNA complexes upon addition of complementary nucleotides and derived rates of conformational changes. We further found that, while incorrect nucleotides are quickly rejected, they nonetheless stabilize the polymerase-DNA complex, suggesting that Pol β, when bound to a lesion, has a strong commitment to nucleotide incorporation and thus repair. In summary, the observation and quantification of fingers movement in human Pol β reported here provide new insights into the delicate mechanisms of prechemistry nucleotide selection.

scholarly journals Probing the stability of the SpCas9-DNA complex after cleavage

2021 ◽  
Author(s):  
Pierre Aldag ◽  
Fabian Welzel ◽  
Leonhard Jakob ◽  
Andreas Schmidbauer ◽  
Marius Rutkauskas ◽  
...  
Keyword(s):  
Single Molecule ◽  
Magnetic Tweezers ◽  
Slowing Down ◽  
Target Strand ◽  
Dna Strands ◽  
Dna Strand ◽  
The Stability ◽  
The Individual ◽  
Dna Complex

CRISPR-Cas9 is a ribonucleoprotein complex that sequence-specifically binds and cleaves double-stranded DNA. Wildtype Cas9 as well as its nickase and cleavage-incompetent mutants have been used in various biological techniques due to their versatility and programmable specificity. Cas9 has been shown to bind very stably to DNA even after cleavage of the individual DNA strands, inhibiting further turnovers and considerably slowing down in-vivo repair processes. This poses an obstacle in genome editing applications. Here, we employed single-molecule magnetic tweezers to investigate the binding stability of different S. pyogenes Cas9 variants after cleavage by challenging them with supercoiling. We find that different release mechanisms occur depending on which DNA strand is cleaved. After non-target strand cleavage, supercoils are immediately but slowly released by swiveling of the non-target strand around the DNA with friction. Consequently, Cas9 and its non-target strand nicking mutant stay stably bound to the DNA for many hours even at elevated torsional stress. After target-strand cleavage, supercoils are only removed after the collapse of the R-loop. We identified several states with different stabilities of the R-loop. Most importantly, we find that the post-cleavage state of Cas9 exhibits a higher stability compared to the pre-cleavage state. This suggests that Cas9 has evolved to remain tightly bound to its cut target.

scholarly journals A Helicase-tethered ORC Flip Enables Bidirectional Helicase Loading

2021 ◽  
Author(s):  
Shalini Gupta ◽  
Larry J. Friedman ◽  
Jeff Gelles ◽  
Stephen P. Bell
Keyword(s):  
Dna Binding ◽  
Binding Site ◽  
Single Molecule ◽  
Rapid Change ◽  
Single Molecule Spectroscopy ◽  
Replication Origins ◽  
Helicase Loading ◽  
Dna Binding Site ◽  
Opposite Face ◽  
Dna Complex

AbstractReplication origins are licensed by loading two Mcm2-7 helicases around DNA in a head-to-head conformation poised to initiate bidirectional replication. This process requires ORC, Cdc6, and Cdt1. Although different Cdc6 and Cdt1 molecules load each helicase, whether two ORC proteins are required is unclear. Using colocalization single-molecule spectroscopy combined with FRET, we investigated interactions between ORC and Mcm2-7 during helicase loading. We demonstrate that a single ORC molecule can recruit both Mcm2-7/Cdt1 complexes via similar interactions that end upon Cdt1 release. Between the first and second helicase recruitment, we observe a rapid change in interactions between ORC and the first Mcm2-7. In quick succession ORC breaks the interactions mediating first Mcm2-7 recruitment, releases from its initial DNA-binding site, and forms a new interaction with the opposite face of the first Mcm2-7. This rearrangement requires release of the first Cdt1 and tethers ORC as it flips over the first Mcm2-7 to form an inverted Mcm2-7-ORC-DNA complex required for second-helicase recruitment. To ensure correct licensing, this complex is maintained until head-to-head interactions between the two helicases are formed. Our findings reconcile previous observations and reveal a highly-coordinated series of events through which a single ORC molecule can load two oppositely-oriented helicases.

Determining the Zero-Force Binding Energetics of an Intercalated DNA Complex by a Single-Molecule Approach

ChemPhysChem ◽  
2009 ◽  
Vol 10 (16) ◽  
pp. 2791-2794 ◽  
Author(s):  
Tzu-Sen Yang ◽  
Yujia Cui ◽  
Chien-Ming Wu ◽  
Jem-Mau Lo ◽  
Chi-Shiun Chiang ◽  
...  
Keyword(s):  
Single Molecule ◽  
Zero Force ◽  
Binding Energetics ◽  
Dna Complex

scholarly journals Single-Molecule Nanopositioning: Structural Transitions of a Helicase-DNA Complex during ATP Hydrolysis

2011 ◽  
Vol 101 (4) ◽  
pp. 976-984 ◽  
Author(s):  
Hamza Balci ◽  
Sinan Arslan ◽  
Sua Myong ◽  
Timothy M. Lohman ◽  
Taekjip Ha
Keyword(s):  
Single Molecule ◽  
Atp Hydrolysis ◽  
Structural Transitions ◽  
Dna Complex

Cationic copolymers nanoparticles for nonviral gene vectors: Synthesis, characterization, and application in gene delivery

2010 ◽  
Vol 9999A ◽  
pp. NA-NA ◽  
Author(s):  
Giovanna Gomez d'Ayala ◽  
Anna Calarco ◽  
Mario Malinconico ◽  
Paola Laurienzo ◽  
Orsolina Petillo ◽  
...  
Keyword(s):  
Gene Delivery ◽  
Gene Vectors ◽  
Cationic Copolymers

scholarly journals Dynamics of Synaptic SfiI-DNA Complex: Single-Molecule Fluorescence Analysis

2007 ◽  
Vol 92 (9) ◽  
pp. 3241-3250 ◽  
Author(s):  
Mikhail A. Karymov ◽  
Alexey V. Krasnoslobodtsev ◽  
Yuri L. Lyubchenko
Keyword(s):  
Single Molecule ◽  
Fluorescence Analysis ◽  
Single Molecule Fluorescence ◽  
Dna Complex

scholarly journals Function of a viral genome packaging motor from bacteriophage T4 is insensitive to DNA sequence

2020 ◽  
Vol 48 (20) ◽  
pp. 11602-11614
Author(s):  
Youbin Mo ◽  
Nicholas Keller ◽  
Damian delToro ◽  
Neeti Ananthaswamy ◽  
Stephen C Harvey ◽  
...  
Keyword(s):  
Dna Sequence ◽  
Motor Function ◽  
Optical Tweezers ◽  
Single Molecule ◽  
Dna Sequences ◽  
Viral Genome ◽  
Bacteriophage T4 ◽  
Repeated Measurements ◽  
Genome Packaging ◽  
Sequence Dependence

Abstract Many viruses employ ATP-powered motors during assembly to translocate DNA into procapsid shells. Previous reports raise the question if motor function is modulated by substrate DNA sequence: (i) the phage T4 motor exhibits large translocation rate fluctuations and pauses and slips; (ii) evidence suggests that the phage phi29 motor contacts DNA bases during translocation; and (iii) one theoretical model, the ‘B-A scrunchworm’, predicts that ‘A-philic’ sequences that transition more easily to A-form would alter motor function. Here, we use single-molecule optical tweezers measurements to compare translocation of phage, plasmid, and synthetic A-philic, GC rich sequences by the T4 motor. We observed no significant differences in motor velocities, even with A-philic sequences predicted to show higher translocation rate at high applied force. We also observed no significant changes in motor pausing and only modest changes in slipping. To more generally test for sequence dependence, we conducted correlation analyses across pairs of packaging events. No significant correlations in packaging rate, pausing or slipping versus sequence position were detected across repeated measurements with several different DNA sequences. These studies suggest that viral genome packaging is insensitive to DNA sequence and fluctuations in packaging motor velocity, pausing and slipping are primarily stochastic temporal events.

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