Faculty Opinions recommendation of Single-molecule analysis reveals that the lagging strand increases replisome processivity but slows replication fork progression.

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.

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

Science Advances ◽

10.1126/sciadv.abc0330 ◽

2020 ◽

Vol 6 (38) ◽

pp. eabc0330 ◽

Cited By ~ 1

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.

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The CMG helicase bypasses DNA protein cross-links to facilitate their repair

10.1101/381582 ◽

2018 ◽

Author(s):

Justin L. Sparks ◽

Alan O. Gao ◽

Markus Räschle ◽

Nicolai B. Larsen ◽

Matthias Mann ◽

...

Keyword(s):

Replication Fork ◽

Dna Helicase ◽

Genome Integrity ◽

Cross Link ◽

Replication Fork Progression ◽

Egg Extracts ◽

Cross Links ◽

Leading Strand ◽

Nucleoprotein Complexes ◽

Lagging Strand

SummaryCovalent and non-covalent nucleoprotein complexes impede replication fork progression and thereby threaten genome integrity. UsingXenopus laevisegg extracts, we previously showed that when a replication fork encounters a covalent DNA-protein cross-link (DPC) on the leading strand template, the DPC is degraded to a short peptide, allowing its bypass by translesion synthesis polymerases. Strikingly, we show here that when DPC proteolysis is blocked, the replicative DNA helicase (CMG), which travels on the leading strand template, still bypasses the intact DPC. The DNA helicase RTEL1 facilitates bypass, apparently by translocating along the lagging strand template and generating single-stranded DNA downstream of the DPC. Remarkably, RTEL1 is required for efficient DPC proteolysis, suggesting that CMG bypass of a DPC normally precedes its proteolysis. RTEL1 also promotes fork progression past non-covalent protein-DNA complexes. Our data suggest a unified model for the replisome’s response to nucleoprotein barriers.

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Replisome bypass of transcription complexes and R-loops

Nucleic Acids Research ◽

10.1093/nar/gkaa741 ◽

2020 ◽

Vol 48 (18) ◽

pp. 10353-10367

Author(s):

Jan-Gert Brüning ◽

Kenneth J Marians

Keyword(s):

Dna Replication ◽

Replication Fork ◽

Genome Instability ◽

Bacterial Dna ◽

Template Strand ◽

Replication Fork Progression ◽

Leading Strand ◽

Fork Stalling ◽

Transcription Complexes ◽

Lagging Strand

Abstract The vast majority of the genome is transcribed by RNA polymerases. G+C-rich regions of the chromosomes and negative superhelicity can promote the invasion of the DNA by RNA to form R-loops, which have been shown to block DNA replication and promote genome instability. However, it is unclear whether the R-loops themselves are sufficient to cause this instability or if additional factors are required. We have investigated replisome collisions with transcription complexes and R-loops using a reconstituted bacterial DNA replication system. RNA polymerase transcription complexes co-directionally oriented with the replication fork were transient blockages, whereas those oriented head-on were severe, stable blockages. On the other hand, replisomes easily bypassed R-loops on either template strand. Replication encounters with R-loops on the leading-strand template (co-directional) resulted in gaps in the nascent leading strand, whereas lagging-strand template R-loops (head-on) had little impact on replication fork progression. We conclude that whereas R-loops alone can act as transient replication blocks, most genome-destabilizing replication fork stalling likely occurs because of proteins bound to the R-loops.

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Mechanisms for Maintaining Eukaryotic Replisome Progression in the Presence of DNA Damage

Frontiers in Molecular Biosciences ◽

10.3389/fmolb.2021.712971 ◽

2021 ◽

Vol 8 ◽

Author(s):

Thomas A. Guilliam

Keyword(s):

Dna Damage ◽

Single Molecule ◽

Replication Fork ◽

Genome Stability ◽

Genome Duplication ◽

Damage Tolerance ◽

Tolerance Mechanisms ◽

Dna Damage Tolerance ◽

Replication Fork Progression ◽

The Impact

The eukaryotic replisome coordinates template unwinding and nascent-strand synthesis to drive DNA replication fork progression and complete efficient genome duplication. During its advancement along the parental template, each replisome may encounter an array of obstacles including damaged and structured DNA that impede its progression and threaten genome stability. A number of mechanisms exist to permit replisomes to overcome such obstacles, maintain their progression, and prevent fork collapse. A combination of recent advances in structural, biochemical, and single-molecule approaches have illuminated the architecture of the replisome during unperturbed replication, rationalised the impact of impediments to fork progression, and enhanced our understanding of DNA damage tolerance mechanisms and their regulation. This review focusses on these studies to provide an updated overview of the mechanisms that support replisomes to maintain their progression on an imperfect template.

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

Molecular and Cellular Biology ◽

10.1128/mcb.00576-18 ◽

2019 ◽

Vol 39 (10) ◽

Cited By ~ 1

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.

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The intersection of DNA replication with antisense 3′ RNA processing in Arabidopsis FLC chromatin silencing

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.2107483118 ◽

2021 ◽

Vol 118 (28) ◽

pp. e2107483118

Author(s):

Colette L. Baxter ◽

Saša Šviković ◽

Julian E. Sale ◽

Caroline Dean ◽

Silvia Costa

Keyword(s):

Single Molecule ◽

Antisense Rna ◽

Replication Fork ◽

Replication Forks ◽

Line System ◽

Replication Fork Progression ◽

Fiber Analysis ◽

Floral Repressor ◽

Processing Factors ◽

Chicken Dt40 Cell

How noncoding transcription influences chromatin states is still unclear. The Arabidopsis floral repressor gene FLC is quantitatively regulated through an antisense-mediated chromatin silencing mechanism. The FLC antisense transcripts form a cotranscriptional R-loop that is dynamically resolved by RNA 3′ processing factors (FCA and FY), and this is linked to chromatin silencing. Here, we investigate this silencing mechanism and show, using single-molecule DNA fiber analysis, that FCA and FY are required for unimpeded replication fork progression across the Arabidopsis genome. We then employ the chicken DT40 cell line system, developed to investigate sequence-dependent replication and chromatin inheritance, and find that FLC R-loop sequences have an orientation-dependent ability to stall replication forks. These data suggest a coordination between RNA 3′ processing of antisense RNA and replication fork progression in the inheritance of chromatin silencing at FLC.

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