Rescuing Replication from Barriers: Mechanistic Insights from Single-Molecule Studies

Sun, Bo

doi:10.1128/mcb.00576-18

Cited by 3 publications

(4 citation statements)

References 80 publications

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“…These results suggested a potential replication recovery pathway in which the inactivation of a replisome by a DNA lesion might also be capable of resuming replication from a stalled RNAP. Recently, Sun et al used an optical tweezer to examine this possibility and showed that T7 helicase associates with nonreplicating DNAP, jointly unwinding DNA at a fork …”

Section: Optical Tweezersmentioning

confidence: 99%

“…Recently, Sun et al used an optical tweezer to examine this possibility and showed that T7 helicase associates with nonreplicating DNAP, jointly unwinding DNA at a fork. 43 By using an optical tweezer coupled to confocal microscopy, Bi et al found that two BLM helicases carrying out the unwinding of two forked DNA substrates oligomerize upon head-on collision leading to fork convergence. 44 Oligomerization is mediated by the helicase and RNaseD C-terminal (HRDC) domain of BLM such that it tightly bridges two newly generated ssDNA strands from unwinding.…”

Section: ■ Optical Tweezersmentioning

confidence: 99%

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Insights into the Dynamics and Helicase Activity of RecD2 of Deinococcus radiodurans during DNA Repair: A Single-Molecule Perspective

2023

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While the double helix is the most stable conformation of DNA inside cells, its transient unwinding and subsequent partial separation of the two complementary strands yields an intermediate single-stranded DNA (ssDNA). The ssDNA is involved in all major DNA transactions such as replication, transcription, recombination, and repair. The process of DNA unwinding and translocation is shouldered by helicases that transduce the chemical energy derived from nucleotide triphosphate (NTP) hydrolysis to mechanical energy and utilize it to destabilize hydrogen bonds between complementary base pairs. Consequently, a comprehensive understanding of the molecular mechanisms of these enzymes is essential. In the last few decades, a combination of single-molecule techniques (force-based manipulation and visualization) have been employed to study helicase action. These approaches have allowed researchers to study the single helicase−DNA complex in real-time and the free energy landscape of their interaction together with the detection of conformational intermediates and molecular heterogeneity during the course of helicase action. Furthermore, the unique ability of these techniques to resolve helicase motion at nanometer and millisecond spatial and temporal resolutions, respectively, provided surprising insights into their mechanism of action. This perspective outlines the contribution of single-molecule methods in deciphering molecular details of helicase activities. It also exemplifies how each technique was or can be used to study the helicase action of RecD2 in recombination DNA repair.

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Section: Optical Tweezersmentioning

confidence: 99%

Section: ■ Optical Tweezersmentioning

confidence: 99%

Insights into the Dynamics and Helicase Activity of RecD2 of Deinococcus radiodurans during DNA Repair: A Single-Molecule Perspective

2023

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“…Compared with ensemble studies, single-molecule approaches have shown great advantages in measurements of molecular heterogeneity, distributions in molecular behaviors, and real-time dynamics of single biomolecules (Cordes et al 2015 ; Cuculis and Schroeder 2017 ; Leake 2013 ; Sun 2019 ; Sun and Wang 2016 ). A wide variety of fluorescence spectroscopy-based (DNA curtains and fluorescence resonance energy transfer) and force spectroscopy-based (magnetic tweezers, optical tweezers, and atomic force microscopy) single-molecule techniques have been used to investigate different aspects of Cas9 proteins (Cuculis and Schroeder 2017 ; Globyte et al 2018 ; Singh and Ha 2018 ; Whinn et al 2019 ).…”

Section: Single-molecule Techniques For Cas9 Studiesmentioning

confidence: 99%

Molecular mechanisms of <i>Streptococcus pyogenes</i> Cas9: a single-molecule perspective

Zhang¹,

Chen²,

Sun³

2021

Biophysics Reports

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Cas9 is an RNA-guided endonuclease from the type II CRISPR-Cas system that employs RNA–DNA base pairing to target and cleave foreign DNA in bacteria. Due to its robust and programmable activity, Cas9 has been repurposed as a revolutionary technology for wide-ranging biological and medical applications. A comprehensive understanding of Cas9 mechanisms at the molecular level would aid in its better usage as a genome tool. Over the past few years, single-molecule techniques, such as fluorescence resonance energy transfer, DNA curtains, magnetic tweezers, and optical tweezers, have been extensively applied to characterize the detailed molecular mechanisms of Cas9 proteins. These techniques allow researchers to monitor molecular dynamics and conformational changes, probe essential DNA–protein interactions, detect intermediate states, and distinguish heterogeneity along the reaction pathway, thus providing enriched functional and mechanistic perspectives. This review outlines the single-molecule techniques that have been utilized for the investigation of Cas9 proteins and discusses insights into the mechanisms of the widely used Streptococcus pyogenes (Sp) Cas9 revealed through these techniques.

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“…This high-powered approach, and other innovative strategies in SM experimentation, ushered in a new era for mechanistic studies of DNA helicases. Some of the important and diverse innovations in SM studies of DNA helicases are discussed below from a historical perspective, but due to the broad scope of the field readers are encouraged to consult some timely reviews [207][208][209].…”

Section: Single-molecule Studies Of Helicase-catalyzed Dna Unwindingmentioning

confidence: 99%

History of DNA Helicases

Brosh

Matson

2020

Genes

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Since the discovery of the DNA double helix, there has been a fascination in understanding the molecular mechanisms and cellular processes that account for: (i) the transmission of genetic information from one generation to the next and (ii) the remarkable stability of the genome. Nucleic acid biologists have endeavored to unravel the mysteries of DNA not only to understand the processes of DNA replication, repair, recombination, and transcription but to also characterize the underlying basis of genetic diseases characterized by chromosomal instability. Perhaps unexpectedly at first, DNA helicases have arisen as a key class of enzymes to study in this latter capacity. From the first discovery of ATP-dependent DNA unwinding enzymes in the mid 1970’s to the burgeoning of helicase-dependent pathways found to be prevalent in all kingdoms of life, the story of scientific discovery in helicase research is rich and informative. Over four decades after their discovery, we take this opportunity to provide a history of DNA helicases. No doubt, many chapters are left to be written. Nonetheless, at this juncture we are privileged to share our perspective on the DNA helicase field – where it has been, its current state, and where it is headed.

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Rescuing Replication from Barriers: Mechanistic Insights from Single-Molecule Studies

Cited by 3 publications

References 80 publications

Insights into the Dynamics and Helicase Activity of RecD2 of Deinococcus radiodurans during DNA Repair: A Single-Molecule Perspective

Insights into the Dynamics and Helicase Activity of RecD2 of Deinococcus radiodurans during DNA Repair: A Single-Molecule Perspective

Molecular mechanisms of <i>Streptococcus pyogenes</i> Cas9: a single-molecule perspective

History of DNA Helicases

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