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

2019 ◽  
Vol 39 (10) ◽  
Author(s):  
Bo Sun
Keyword(s):  
Real Time ◽  
Optical Tweezers ◽  
Single Molecule ◽  
Replication Fork ◽  
Magnetic Tweezers ◽  
Origin Of Replication ◽  
Replication Fork Progression ◽  
Nucleotide Incorporation ◽  
Single Molecule Studies ◽  
Replication Failure

ABSTRACT To prevent replication failure due to fork barriers, several mechanisms have evolved to restart arrested forks independent of the origin of replication. Our understanding of these mechanisms that underlie replication reactivation has been aided through unique dynamic perspectives offered by single-molecule techniques. These techniques, such as optical tweezers, magnetic tweezers, and fluorescence-based methods, allow researchers to monitor the unwinding of DNA by helicase, nucleotide incorporation during polymerase synthesis, and replication fork progression in real time. In addition, they offer the ability to distinguish DNA intermediates after obstacles to replication at high spatial and temporal resolutions, providing new insights into the replication reactivation mechanisms. These and other highlights of single-molecule techniques and remarkable studies on the recovery of the replication fork from barriers will be discussed in this review.

scholarly journals Single-molecule imaging reveals control of parental histone recycling by free histones during DNA replication

2020 ◽  
Vol 6 (38) ◽  
pp. eabc0330 ◽  
Author(s):  
D. T. Gruszka ◽  
S. Xie ◽  
H. Kimura ◽  
H. Yardimci
Keyword(s):  
Dna Replication ◽  
Real Time ◽  
Single Molecule ◽  
Replication Fork ◽  
Epigenetic Inheritance ◽  
Replication Forks ◽  
Replication Fork Progression ◽  
Egg Extracts ◽  
Fork Stalling ◽  
The Moment

During replication, nucleosomes are disrupted ahead of the replication fork, followed by their reassembly on daughter strands from the pool of recycled parental and new histones. However, because no previous studies have managed to capture the moment that replication forks encounter nucleosomes, the mechanism of recycling has remained unclear. Here, through real-time single-molecule visualization of replication fork progression in Xenopus egg extracts, we determine explicitly the outcome of fork collisions with nucleosomes. Most of the parental histones are evicted from the DNA, with histone recycling, nucleosome sliding, and replication fork stalling also occurring but at lower frequencies. Critically, we find that local histone recycling becomes dominant upon depletion of endogenous histones from extracts, revealing that free histone concentration is a key modulator of parental histone dynamics at the replication fork. The mechanistic details revealed by these studies have major implications for our understanding of epigenetic inheritance.

scholarly journals Single-molecule imaging of DNA gyrase activity in livingEscherichia coli

2018 ◽  
Author(s):  
Mathew Stracy ◽  
Adam J.M. Wollman ◽  
Elzbieta Kaja ◽  
Jacek Gapinski ◽  
Ji-Eun Lee ◽  
...  
Keyword(s):  
Single Molecule ◽  
High Speed ◽  
Replication Fork ◽  
Dna Gyrase ◽  
Chromosomal Dna ◽  
Replication Fork Progression ◽  
Dna Strands ◽  
Steady State Levels ◽  
Negative Supercoiling ◽  
Supercoil Relaxation

ABSTRACTBacterial DNA gyrase introduces negative supercoils into chromosomal DNA and relaxes positive supercoils introduced by replication and transiently by transcription. Removal of these positive supercoils is essential for replication fork progression and for the overall unlinking of the two duplex DNA strands, as well as for ongoing transcription. To address how gyrase copes with these topological challenges, we used high-speed single-molecule fluorescence imaging in liveEscherichia colicells. We demonstrate that at least 300 gyrase molecules are stably bound to the chromosome at any time, with ∼12 enzymes enriched near each replication fork. Trapping of reaction intermediates with ciprofloxacin revealed complexes undergoing catalysis. Dwell times of ∼2 s were observed for the dispersed gyrase molecules, which we propose maintain steady-state levels of negative supercoiling of the chromosome. In contrast, the dwell time of replisome-proximal molecules was ∼8 s, consistent with these catalyzing processive positive supercoil relaxation in front of the progressing replisome.

DNA visualization in single molecule studies carried out with optical tweezers: Covalent versus non-covalent attachment of fluorophores

2015 ◽  
Vol 466 (2) ◽  
pp. 226-231 ◽  
Author(s):  
Sandy Suei ◽  
Allan Raudsepp ◽  
Lisa M. Kent ◽  
Stephen A.J. Keen ◽  
Vyacheslav V. Filichev ◽  
...  
Keyword(s):  
Optical Tweezers ◽  
Single Molecule ◽  
Covalent Attachment ◽  
Single Molecule Studies

scholarly journals NAP1-Assisted Nucleosome Assembly on DNA Measured in Real Time by Single-Molecule Magnetic Tweezers

PLoS ONE ◽  
2012 ◽  
Vol 7 (9) ◽  
pp. e46306 ◽  
Author(s):  
Rifka Vlijm ◽  
Jeremy S. J. Smitshuijzen ◽  
Alexandra Lusser ◽  
Cees Dekker
Keyword(s):  
Real Time ◽  
Single Molecule ◽  
Magnetic Tweezers ◽  
Nucleosome Assembly

scholarly journals Disturbance-free rapid solution exchange for magnetic tweezers single-molecule studies

2015 ◽  
Vol 43 (17) ◽  
pp. e113-e113 ◽  
Author(s):  
Shimin Le ◽  
Mingxi Yao ◽  
Jin Chen ◽  
Artem K. Efremov ◽  
Sara Azimi ◽  
...  
Keyword(s):  
Single Molecule ◽  
Magnetic Tweezers ◽  
Solution Exchange ◽  
Single Molecule Studies

scholarly journals Single-Molecule Insights Into the Dynamics of Replicative Helicases

2021 ◽  
Vol 8 ◽  
Author(s):  
Richard R. Spinks ◽  
Lisanne M. Spenkelink ◽  
Nicholas E. Dixon ◽  
Antoine M. van Oijen
Keyword(s):  
Single Molecule ◽  
Molecular Motors ◽  
Replication Fork ◽  
Magnetic Tweezers ◽  
Replication Initiation ◽  
Model Systems ◽  
Replication Forks ◽  
Nucleotide Hydrolysis ◽  
Replicative Polymerases ◽  
Replication Factors

Helicases are molecular motors that translocate along single-stranded DNA and unwind duplex DNA. They rely on the consumption of chemical energy from nucleotide hydrolysis to drive their translocation. Specialized helicases play a critically important role in DNA replication by unwinding DNA at the front of the replication fork. The replicative helicases of the model systems bacteriophages T4 and T7, Escherichia coli and Saccharomyces cerevisiae have been extensively studied and characterized using biochemical methods. While powerful, their averaging over ensembles of molecules and reactions makes it challenging to uncover information related to intermediate states in the unwinding process and the dynamic helicase interactions within the replisome. Here, we describe single-molecule methods that have been developed in the last few decades and discuss the new details that these methods have revealed about replicative helicases. Applying methods such as FRET and optical and magnetic tweezers to individual helicases have made it possible to access the mechanistic aspects of unwinding. It is from these methods that we understand that the replicative helicases studied so far actively translocate and then passively unwind DNA, and that these hexameric enzymes must efficiently coordinate the stepping action of their subunits to achieve unwinding, where the size of each step is prone to variation. Single-molecule fluorescence microscopy methods have made it possible to visualize replicative helicases acting at replication forks and quantify their dynamics using multi-color colocalization, FRAP and FLIP. These fluorescence methods have made it possible to visualize helicases in replication initiation and dissect this intricate protein-assembly process. In a similar manner, single-molecule visualization of fluorescent replicative helicases acting in replication identified that, in contrast to the replicative polymerases, the helicase does not exchange. Instead, the replicative helicase acts as the stable component that serves to anchor the other replication factors to the replisome.

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