scholarly journals Elastic Coupling Between RNA Degradation and Unwinding by an Exoribonuclease

Science ◽  
2012 ◽  
Vol 336 (6089) ◽  
pp. 1726-1729 ◽  
Author(s):  
Gwangrog Lee ◽  
Matthew A. Bratkowski ◽  
Fang Ding ◽  
Ailong Ke ◽  
Taekjip Ha
Keyword(s):  
Single Molecule ◽  
Fluorescence Analysis ◽  
Rna Degradation ◽  
Step Size ◽  
Base Pairs ◽  
Single Nucleotide ◽  
Duplex Stability ◽  
Single Base Pair ◽  
Rna Complex ◽  
Exosome Complex

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 nucleotide step hydrolysis reactions until about four base pairs are unwound in a burst. Kinetic analyses showed that RNA unwinding, not cleavage or strand release, determines the overall RNA degradation rate and that the unwinding step size is determined by the nonlinear elasticity of the Rrp44/RNA complex, but not by duplex stability.

scholarly journals Hexameric helicase G40P unwinds DNA in single base pair steps

eLife ◽  
2019 ◽  
Vol 8 ◽  
Author(s):  
Michael Schlierf ◽  
Ganggang Wang ◽  
Xiaojiang S Chen ◽  
Taekjip Ha
Keyword(s):  
Single Molecule ◽  
Base Pair ◽  
Atp Hydrolysis ◽  
Thermal Fluctuation ◽  
Dna Unwinding ◽  
Single Molecule Fluorescence ◽  
Step Size ◽  
Structural Investigations ◽  
Single Base ◽  
Single Base Pair

Most replicative helicases are hexameric, ring-shaped motor proteins that translocate on and unwind DNA. Despite extensive biochemical and structural investigations, how their translocation activity is utilized chemo-mechanically in DNA unwinding is poorly understood. We examined DNA unwinding by G40P, a DnaB-family helicase, using a single-molecule fluorescence assay with a single base pair resolution. The high-resolution assay revealed that G40P by itself is a very weak helicase that stalls at barriers as small as a single GC base pair and unwinds DNA with the step size of a single base pair. Binding of a single ATPγS could stall unwinding, demonstrating highly coordinated ATP hydrolysis between six identical subunits. We observed frequent slippage of the helicase, which is fully suppressed by the primase DnaG. We anticipate that these findings allow a better understanding on the fine balance of thermal fluctuation activation and energy derived from hydrolysis.

scholarly journals Hexameric helicase G40P unwinds DNA in single base pair steps

2018 ◽  
Author(s):  
Michael Schlierf ◽  
Ganggang Wang ◽  
Xiaojiang S. Chen ◽  
Taekjip Ha
Keyword(s):  
Single Molecule ◽  
Base Pair ◽  
Atp Hydrolysis ◽  
Thermal Fluctuation ◽  
Dna Unwinding ◽  
Single Molecule Fluorescence ◽  
Step Size ◽  
Structural Investigations ◽  
Single Base ◽  
Single Base Pair

AbstractMost replicative helicases are hexameric, ring-shaped motor proteins that translocate on and unwind DNA. Despite extensive biochemical and structural investigations, how their translocation activity is utilized chemo-mechanically in DNA unwinding is poorly understood. We examined DNA unwinding by G40P, a DnaB-family helicase, using a single-molecule fluorescence assay with a single base pair resolution. The high-resolution assay revealed that G40P by itself is a very weak helicase that stalls at barriers as small as a single GC base pair and unwinds DNA with the step size of a single base pair. Single ATPγS binding could stall unwinding, demonstrating highly coordinated ATP hydrolysis between the six identical subunits. We observed frequent slippage of the helicase, which is fully suppressed by the primase DnaG. We anticipate that these findings allow a better understanding on the fine balance of thermal fluctuation activation and energy derived from hydrolysis.

scholarly journals Highly specific imaging of mRNA in single cells by target RNA-initiated rolling circle amplification

2017 ◽  
Vol 8 (5) ◽  
pp. 3668-3675 ◽  
Author(s):  
Ruijie Deng ◽  
Kaixiang Zhang ◽  
Yupeng Sun ◽  
Xiaojun Ren ◽  
Jinghong Li
Keyword(s):  
Single Molecule ◽  
Single Cells ◽  
Rolling Circle Amplification ◽  
Robust Method ◽  
Single Nucleotide ◽  
Rolling Circle ◽  
Target Rna

We report a robust method for the efficient imaging of mRNA with single-nucleotide and near-single-molecule resolution in single cells.

scholarly journals Single–motor mechanics and models of the myosin motor

2000 ◽  
Vol 355 (1396) ◽  
pp. 441-447 ◽  
Author(s):  
T. Yanagida ◽  
S. Esaki ◽  
A. Hikikoshi Iwane ◽  
Y. Inoue ◽  
A. Ishijima ◽  
...  
Keyword(s):  
Single Molecule ◽  
Atp Hydrolysis ◽  
Accurate Determination ◽  
Step Size ◽  
Single Molecule Level ◽  
Detection Techniques ◽  
Myosin Motor ◽  
Mechanochemical Coupling ◽  
Chemical Events

Recent progress in single–molecule detection techniques is remarkable. These techniques have allowed the accurate determination of myosin–head–induced displacements and how mechanical cycles are coupled to ATP hydrolysis, by measuring individual mechanical events and chemical events of actomyosin directly at the single–molecule level. Here we review our recent work in which we have made detailed measurements of myosin step size and mechanochemical coupling, and propose a model of the myosin motor.

scholarly journals Exosome complex genes mediate RNA degradation and predict survival in mantle cell lymphoma

2019 ◽  
Author(s):  
Weilong Zhang ◽  
Junyong Zhu ◽  
Xue He ◽  
Xiaoni Liu ◽  
Jinhang Li ◽  
...  
Keyword(s):  
Mantle Cell Lymphoma ◽  
Cell Lymphoma ◽  
Rna Degradation ◽  
Mantle Cell ◽  
Exosome Complex

scholarly journals Evolutionary origins of the emergent ST796 clone of vancomycin resistant Enterococcus faecium

2016 ◽  
Author(s):  
Andrew H Buultjens ◽  
Margaret M C Lam ◽  
Susan Ballard ◽  
Ian R Monk ◽  
Andrew A Mahony ◽  
...  
Keyword(s):  
Single Molecule ◽  
Enterococcus Faecium ◽  
Genomic Islands ◽  
Nucleotide Polymorphisms ◽  
Circular Chromosome ◽  
Single Nucleotide ◽  
Evolutionary Origins ◽  
Eastern Australia ◽  
Healthcare Environments ◽  
Vancomycin Resistant

From early 2012, a novel clone of vancomycin resistant Enterococcus faecium (assigned the multi locus sequence type ST796) was simultaneously isolated from geographically separate hospitals in south eastern Australia and New Zealand. Here we describe the complete genome sequence of Ef_aus0233, a representative ST796 E. faecium isolate. We used PacBio single molecule real-time sequencing to establish a high quality, fully assembled genome comprising a circular chromosome of 2,888,087 bp and five plasmids. Comparison of Ef_aus0233 to other E. faecium genomes shows Ef_aus0233 is a member of the epidemic hospital-adapted lineage and has evolved from an ST555-like ancestral progenitor by the accumulation or modification of five mosaic plasmids and five putative prophage, acquisition of two cryptic genomic islands, accrued chromosomal single nucleotide polymorphisms and a 80kb region of recombination, also gaining Tn1549 and Tn916, transposons conferring resistance to vancomycin and tetracycline respectively. The genomic dissection of this new clone presented here underscores the propensity of the hospital E. faecium lineage to change, presumably in response to the specialized conditions of hospital and healthcare environments.

scholarly journals Kinetic and structural mechanism for DNA unwinding by a non-hexameric helicase

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Sean P. Carney ◽  
Wen Ma ◽  
Kevin D. Whitley ◽  
Haifeng Jia ◽  
Timothy M. Lohman ◽  
...  
Keyword(s):  
Optical Tweezers ◽  
Single Molecule ◽  
Atp Hydrolysis ◽  
Variable Number ◽  
Structural Basis ◽  
Variable Step Size ◽  
Dna Unwinding ◽  
Step Size ◽  
Structural Mechanism ◽  
Dynamics Simulations

AbstractUvrD, a model for non-hexameric Superfamily 1 helicases, utilizes ATP hydrolysis to translocate stepwise along single-stranded DNA and unwind the duplex. Previous estimates of its step size have been indirect, and a consensus on its stepping mechanism is lacking. To dissect the mechanism underlying DNA unwinding, we use optical tweezers to measure directly the stepping behavior of UvrD as it processes a DNA hairpin and show that UvrD exhibits a variable step size averaging ~3 base pairs. Analyzing stepping kinetics across ATP reveals the type and number of catalytic events that occur with different step sizes. These single-molecule data reveal a mechanism in which UvrD moves one base pair at a time but sequesters the nascent single strands, releasing them non-uniformly after a variable number of catalytic cycles. Molecular dynamics simulations point to a structural basis for this behavior, identifying the protein-DNA interactions responsible for strand sequestration. Based on structural and sequence alignment data, we propose that this stepping mechanism may be conserved among other non-hexameric helicases.

scholarly journals Somatic mutation landscapes at single-molecule resolution

2021 ◽  
Author(s):  
Stefanie V. Lensing ◽  
Peter Ellis ◽  
Federico Abascal ◽  
Iñigo Martincorena ◽  
Robert J. Osborne
Keyword(s):  
Single Molecule ◽  
Single Cells ◽  
Error Rates ◽  
Dna Molecules ◽  
Base Pairs ◽  
Single Dna Molecules ◽  
Sequencing Library ◽  
Somatic Mutagenesis ◽  
Qpcr Quantification ◽  
Duplex Sequencing

Abstract Somatic mutations drive cancer development and may contribute to ageing and other diseases. Yet, the difficulty of detecting mutations present only in single cells or small clones has limited our knowledge of somatic mutagenesis to a minority of tissues. To overcome these limitations, we introduce nanorate sequencing (NanoSeq), a new duplex sequencing protocol with error rates <5 errors per billion base pairs in single DNA molecules from cell populations. The version of the protocol described here uses clean genome fragmentation with a restriction enzyme to prevent end-repair-associated errors and ddBTPs/dATPs during A-tailing to prevent nick extension. Both changes reduce the error rate of standard duplex sequencing protocols by preventing the fixation of DNA damage into both strands of DNA molecules during library preparation. We also use qPCR quantification of the library prior to amplification to optimise the complexity of the sequencing library given the desired sequencing coverage, maximising duplex coverage. The sample preparation protocol takes between 1 and 2 days, depending on the number of samples processed. The bioinformatic protocol is described in:https://github.com/cancerit/NanoSeqhttps://github.com/fa8sanger/NanoSeq_Paper_Code

scholarly journals Single Molecule Investigation of Ag+ Interactions with Single Cytosine-, Methylcytosine- and Hydroxymethylcytosine-Cytosine Mismatches in a Nanopore

2014 ◽  
Vol 4 (1) ◽  
Author(s):  
Yong Wang ◽  
Bin-Quan Luan ◽  
Zhiyu Yang ◽  
Xinyue Zhang ◽  
Brandon Ritzo ◽  
...  
Keyword(s):  
Hydrogen Bond ◽  
Binding Site ◽  
Single Molecule ◽  
Metal Ion ◽  
Detection Method ◽  
Direct Detection ◽  
Alpha Hemolysin ◽  
Duplex Stability ◽  
Dynamics Simulations ◽  
Direct Detection Method

Abstract Both cytosine-Ag-cytosine interactions and cytosine modifications in a DNA duplex have attracted great interest for research. Cytosine (C) modifications such as methylcytosine (mC) and hydroxymethylcytosine (hmC) are associated with tumorigenesis. However, a method for directly discriminating C, mC and hmC bases without labeling, modification and amplification is still missing. Additionally, the nature of coordination of Ag+ with cytosine-cytosine (C-C) mismatches is not clearly understood. Utilizing the alpha-hemolysin nanopore, we show that in the presence of Ag+, duplex stability is most increased for the cytosine-cytosine (C-C) pair, followed by the cytosine-methylcytosine (C-mC) pair and the cytosine-hydroxymethylcytosine (C-hmC) pair, which has no observable Ag+ induced stabilization. Molecular dynamics simulations reveal that the hydrogen-bond-mediated paring of a C-C mismatch results in a binding site for Ag+. Cytosine modifications (such as mC and hmC) disrupted the hydrogen bond, resulting in disruption of the Ag+ binding site. Our experimental method provides a novel platform to study the metal ion-DNA interactions and could also serve as a direct detection method for nucleobase modifications.

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