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 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.

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

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 Mechanism of duplex unwinding by coronavirus nsp13 helicases

2020 ◽  
Author(s):  
Xiao Hu ◽  
Wei Hao ◽  
Bo Qin ◽  
Zhiqi Tian ◽  
Ziheng Li ◽  
...  
Keyword(s):  
Amino Acid ◽  
Single Molecule ◽  
Hinge Region ◽  
Single Amino Acid ◽  
Amino Acid Difference ◽  
List Type ◽  
Single Molecule Fret ◽  
The Difference ◽  
Dna Complex ◽  
Loaded Mechanism

AbstractThe current COVID-19 pandemic urges in-depth investigation into proteins encoded with coronavirus (CoV), especially conserved CoV replicases. The nsp13 of highly pathogenic MERS-CoV, SARS-CoV-2, and SARS-CoV exhibit the most conserved CoV replicases. Using single-molecule FRET, we observed that MERS-CoV nsp13 unwound DNA in discrete steps of approximately 9 bp when ATP was used. If another NTP was used, then the steps were only 4 to 5 bp. In dwell time analysis, we detected 3 or 4 hidden steps in each unwinding process, which indicated the hydrolysis of 3 or 4 dTTP. Based on crystallographic and biochemical studies of CoV nsp13 helicases, we modeled an unwinding mechanism similar to the spring-loaded mechanism of HCV NS3 helicase, although our model proposes that flexible 1B and stalk domains, by allowing a lag greater than 4 bp during unwinding, cause the accumulated tension on the nsp13-DNA complex. The hinge region between two RecA-like domains in SARS-CoV-2 nsp13 is intrinsically more flexible than in MERS-CoV nsp13 due to the difference of a single amino acid, which causes the former to induce significantly greater NTP hydrolysis. Our findings thus establish a blueprint for determining the unwinding mechanism of a unique helicase family.When dTTP was used as the energy source, 4 hidden steps in each individual unwinding step after 3 - 4 NTP hydrolysis were observed.An unwinding model of MERS-CoV-nsp13 which is similar to the spring-loaded mechanism of HCV NS3 helicase, except the accumulation of tension on nsp13/DNA complex is caused by the flexible 1B and stalk domains that allow a lag of 4-bp in unwinding.Comparing to MERS-CoV nsp13, the hinge region between two RecA-like domains in SARS-CoV-2 nsp13 is intrinsically more flexible due to a single amino acid difference, which contributes to the significantly higher NTP hydrolysis by SARS-CoV-2 nsp13.

Quantifying force-dependent and zero-force DNA intercalation by single-molecule stretching

2007 ◽  
Vol 4 (6) ◽  
pp. 517-522 ◽  
Author(s):  
Ioana D Vladescu ◽  
Micah J McCauley ◽  
Megan E Nuñez ◽  
Ioulia Rouzina ◽  
Mark C Williams
Keyword(s):  
Single Molecule ◽  
Dna Intercalation ◽  
Zero Force

DNA polymerase activity at the single-molecule level

2011 ◽  
Vol 39 (2) ◽  
pp. 595-599 ◽  
Author(s):  
Joshua P. Gill ◽  
Jun Wang ◽  
David P. Millar
Keyword(s):  
Single Molecule ◽  
Conformational Changes ◽  
Protein Complexes ◽  
Dna Polymerases ◽  
Polymerase Activity ◽  
Single Molecule Level ◽  
Nucleotide Incorporation ◽  
Single Molecule Fluorescence Spectroscopy ◽  
Conformational Rearrangements ◽  
Dna Complex

DNA polymerases are essential enzymes responsible for replication and repair of DNA in all organisms. To replicate DNA with high fidelity, DNA polymerases must select the correct incoming nucleotide substrate during each cycle of nucleotide incorporation, in accordance with the templating base. When an incorrect nucleotide is sometimes inserted, the polymerase uses a separate 3′→5′ exonuclease to remove the misincorporated base (proofreading). Large conformational rearrangements of the polymerase–DNA complex occur during both the nucleotide incorporation and proofreading steps. Single-molecule fluorescence spectroscopy provides a unique tool for observation of these dynamic conformational changes in real-time, without the need to synchronize a population of DNA–protein complexes.

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.

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