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Rrp44 (Dis3) is a key catalytic subunit of the yeast exosome complex and can processively digest structured RNA one nucleotide at a time in the 3' to 5' direction. Its motor function is powered by the energy released from the hydrolytic nuclease reaction instead of adenosine triphosphate hydrolysis as in conventional helicases. Single-molecule fluorescence analysis revealed that instead of unwinding RNA in single base pair steps, Rrp44 accumulates the energy released by multiple single nucleotid

RNase H is involved in fundamental cellular processes and is responsible for removing the short stretch of RNA from Okazaki fragments and the long stretch of RNA from R-loops. Defects in RNase H lead to embryo lethality in mice and Aicardi-Goutieres syndrome in humans, suggesting the importance of RNase H. To date, RNase H is known to be a non-sequence-specific endonuclease, but it is not known whether it performs other functions on the structural variants of RNA:DNA hybrids. Here, we used Esche

Bacterial exonuclease III (ExoIII) is a multifunctional enzyme that uses a single active site to perform two conspicuous activities: (i) apurinic/apyrimidinic (AP)-endonuclease and (ii) 3'→5' exonuclease activities. The AP endonuclease activity results in AP site incision, while the exonuclease activity results in the continuous excision of 3' terminal nucleobases to generate a partial duplex for recruiting the downstream DNA polymerase during the base excision repair process (BER). The key dete

Abstract Metal ions at the active site of an enzyme act as cofactors, and their dynamic fluctuations can potentially influence enzyme activity. Here, we use λ-exonuclease as a model enzyme with two Mg 2+ binding sites and probe activity at various concentrations of magnesium by single-molecule-FRET. We find that while Mg A 2+ and Mg B 2+ have similar binding constants, the dissociation rate of Mg A 2+ is two order of magnitude lower than that of Mg B 2+ due to a kinetic-barrier-difference. At ph

During base excision repair, a transient single-stranded DNA (ssDNA) gap is produced at the apurinic/apyrimidinic (AP) site. Exonuclease III, capable of performing both AP endonuclease and exonuclease activity, are responsible for gap creation in bacteria. We used single-molecule fluorescence resonance energy transfer to examine the mechanism of gap creation. We found an AP site anchor-based mechanism by which the intrinsically distributive enzyme binds strongly to the AP site and becomes a proc

Single molecules of beta-1 --> 6-linked d-glucose polysaccharides, when stretched in an atomic force microscope, display a hookean-like elasticity unusual for polymers. High-level ab initio calculations and microsecond-scale molecular dynamics simulations reveal that this elasticity is governed by force-induced rotations of the exocyclic group on the glucopyranose rings from their short and less energetic gt and gg conformations to the extended and high-energy tg state. These observations indica

Phosphates along the DNA function as chemical energy frequently used by nucleases to drive their enzymatic reactions. Exonuclease functions as a machine that converts chemical energy of the phosphodiester-chain into mechanical work. However, the roles of phosphates during exonuclease activities are unknown. We employed λ exonuclease as a model system and investigated the roles of phosphates during degradation via single-molecule fluorescence resonance energy transfer (FRET). We found that 5' pho

Molecular motors have inspired many avenues of research for nanotechnology but most molecular motors studied so far allow only unidirectional movement. The archaeal RNA-exosome is a reversible motor that can either polymerize or degrade an RNA strand, depending on the chemical environments. We developed a single molecule fluorescence assay to analyze the real time locomotion of this nanomachine on RNA. Despite the multimeric structure, the enzyme followed the Michaelis-Menten kinetics with the m

Doing damage to DNA: The effects of UV radiation on the mechanics of individual DNA duplexes (see picture) are revealed by AFM-based spectroscopy. It is found that the width of the characteristic B–S plateau in the force spectrogram of irradiated DNA shortens in a UV-dose-dependent manner. These mechanical effects likely represent the local unwinding of the double helix caused by a massive formation of pyrimidine dimers and 6–4 lesions.

The determination of the dissociation constant (K_d) is pivotal in biochemistry and pharmacology for understanding binding affinities in chemical reactions, which is crucial for drug development and comprehending biological systems. Here, we introduce a single-molecule fluorescence resonance energy transfer-based method for determining K_d, alongside the conventional electrophoretic mobility shift assay method of K_d, offering insights into thermodynamic interaction

ADVERTISEMENT RETURN TO ISSUEPREVAddition/CorrectionORIGINAL ARTICLEThis notice is a correctionCorrection to Reversible and Controllable Nanolocomotion of an RNA-Processing MachineryGwangrog Lee, Sophia Hartung, Karl-Peter Hopfner*, and Taekjip Ha*Cite this: Nano Lett. 2013, 13, 4, 1867Publication Date (Web):March 22, 2013Publication History Published online22 March 2013Published inissue 10 April 2013https://pubs.acs.org/doi/10.1021/nl401025thttps://doi.org/10.1021/nl401025tcorrectionACS Publica

The exposure of cancer cells to ionizing radiation results in potentially lethal DNA lesions. For this reason, identification and quantification of various lesions have intensively been investigated. It has also been anticipated that DNA lesions may affect not only the chemical but also the mechanical integrity of the double helix. However, the relationship between DNA damage and mechanics has not been studied. Here, the mechanical properties of DNA damaged by ionizing radiation are examined at