What is all this fuss about Tus? Comparison of recent findings from biophysical and biochemical experiments

Berghuis, Bojk A.; Raducanu, Vlad-Stefan; Elshenawy, Mohamed M.; Jergic, Slobodan; Depken, Martin; Dixon, Nicholas E.; Hamdan, Samir M.; Dekker, Nynke H.

doi:10.1080/10409238.2017.1394264

Cited by 16 publications

(21 citation statements)

References 28 publications

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“…Fork blockage is mediated by a ‘molecular mouse trap mechanism’ in which a cytosine in the Ter site flips into a specific pocket on Tus during strand separation as the oncoming helicases approach the non-permissive face of the complex 5 . This locking mechanism was recently discussed in the light of single molecule studies 3 . Unlike the Tus-Ter locking mechanism described in E. coli , the mechanisms underlying replication fork stalling by barrier proteins bound to specific sites in eukaryotic genomes remain unknown.…”

Section: Introductionmentioning

confidence: 88%

“…Nonetheless, programmed fork stalling occurs at specific sites in genomes mediated by the interaction of these sites with transacting barrier proteins 2 . A well-studied example of this mechanism is the Tus-Ter complex in E. coli 3 . E. coli is able to minimize collisions between DNA and RNA polymerases as its circular DNA is being replicated by oppositely oriented forks with fork barrier sites in the terminus region of its genome, such that the most active genes are replicated and transcribed in the same direction 4 .…”

Section: Introductionmentioning

confidence: 99%

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Structure of the replication regulator Sap1 reveals functionally important interfaces

Jørgensen

Ekundayo

Zaratiegui

et al. 2018

Sci Rep

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The mechanism by which specific protein-DNA complexes induce programmed replication fork stalling in the eukaryotic genome remains poorly understood. In order to shed light on this process we carried out structural investigations on the essential fission yeast protein Sap1. Sap1 was identified as a protein involved in mating-type switching in Schizosaccharomyces pombe, and has been shown to be involved in programmed replication fork stalling. Interestingly, Sap1 assumes two different DNA binding modes. At the mating-type locus dimers of Sap1 bind the SAS1 sequence in a head-to-head arrangement, while they bind to replication fork blocking sites at rDNA and Tf2 transposons in a head-to-tail mode. In this study, we have solved the crystal structure of the Sap1 DNA binding domain and we observe that Sap1 molecules interact in the crystal using a head-to-tail arrangement that is compatible with DNA binding. We find that Sap1 mutations which alleviate replication-fork blockage at Tf2 transposons in CENP-B mutants map to the head-to-tail interface. Furthermore, several other mutations introduced in this interface are found to be lethal. Our data suggests that essential functions of Sap1 depend on its head-to-tail oligomerization.

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Section: Introductionmentioning

confidence: 88%

Section: Introductionmentioning

confidence: 99%

Structure of the replication regulator Sap1 reveals functionally important interfaces

Jørgensen

Ekundayo

Zaratiegui

et al. 2018

Sci Rep

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show abstract

“…However, given the possibility of polymerase-helicase uncoupling (Katou et al, 2003;Pacek et al, 2006;Graham et al, 2017) one has to be cautious about inferring replisome position (a protein-based measure) from the fork position (DNA-based), and vice versa. Recent singlemolecule replication (Sparks et al, 2019) and DNA unwinding (Berghuis et al, 2018) imaging methods allow for combined detection of protein and DNA chromatin components, and introduction of a labeled barrier [such as Cas9, (Vrtis et al, 2021)] helps one to focus on events around it. Along the same lines, the field would benefit from the development of new methods capable of simultaneous co-detection of replisome proteins and nascent fork DNA components at singlenucleotide resolution.…”

Section: Approaches To Study Fork Progressionmentioning

confidence: 99%

Approaching Protein Barriers: Emerging Mechanisms of Replication Pausing in Eukaryotes

Shyian

Shore

2021

Front. Cell Dev. Biol.

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During nuclear DNA replication multiprotein replisome machines have to jointly traverse and duplicate the total length of each chromosome during each cell cycle. At certain genomic locations replisomes encounter tight DNA-protein complexes and slow down. This fork pausing is an active process involving recognition of a protein barrier by the approaching replisome via an evolutionarily conserved Fork Pausing/Protection Complex (FPC). Action of the FPC protects forks from collapse at both programmed and accidental protein barriers, thus promoting genome integrity. In addition, FPC stimulates the DNA replication checkpoint and regulates topological transitions near the replication fork. Eukaryotic cells have been proposed to employ physiological programmed fork pausing for various purposes, such as maintaining copy number at repetitive loci, precluding replication-transcription encounters, regulating kinetochore assembly, or controlling gene conversion events during mating-type switching. Here we review the growing number of approaches used to study replication pausing in vivo and in vitro as well as the characterization of additional factors recently reported to modulate fork pausing in different systems. Specifically, we focus on the positive role of topoisomerases in fork pausing. We describe a model where replisome progression is inherently cautious, which ensures general preservation of fork stability and genome integrity but can also carry out specialized functions at certain loci. Furthermore, we highlight classical and novel outstanding questions in the field and propose venues for addressing them. Given how little is known about replisome pausing at protein barriers in human cells more studies are required to address how conserved these mechanisms are.

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“…The ability to carry out the convergence and fusion of replication fork complexes with high accuracy is essential to the maintenance of genomic stability and cell survival (3). Recent studies in both prokaryotes and eukaryotes have highlighted the fact that the fusion of replication fork complexes requires careful orchestration by a variety of protein co-factors (1,(3)(4)(5)(6)(7).…”

Section: Introductionmentioning

confidence: 99%

“…The asymmetry of replisome arrest at the Tus- ter complex has been extensively studied (5, 11). These studies have demonstrated that polar arrest at Tus is triggered by the approaching replisome unwinding the double-stranded DNA immediately adjacent to the non-permissive face (12).…”

Section: Introductionmentioning

confidence: 99%

Termination of DNA replication at Tus-terbarriers results in under-replication of template DNA

Jameson

Rudolph

Hawkins

2021

Preprint

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The complete and accurate duplication of genomic information is vital to maintain genome stability in all domains of life. In Escherichia coli, replication termination, the final stage of the duplication process, is confined to the replication fork trap region by multiple unidirectional fork barriers formed by the binding of Tus protein to genomic ter sites. Termination typically occurs away from Tus-ter complexes, but they become part of the fork fusion process when a delay to one replisome allows the second to travel more than halfway around the chromosome. In this instance, replisome progression is blocked at the non-permissive interface of Tus-ter and termination occurs when a converging replisome meets the non-permissive interface. To investigate the consequences of replication fork fusion at Tus-ter complexes, we established a plasmid-based replication system where we could mimic the termination process at Tus-ter in vitro. We developed a termination mapping assay to measure leading strand replication fork progression and demonstrate that the DNA template is under-replicated by 1524 bases when replication forks fuse at Tus-ter complexes. This gap could not be closed by the inclusion of lagging strand processing enzymes as well as several helicases that promote DNA replication. Our results indicate that accurate fork fusion at Tus-ter barriers requires further enzymatic processing, highlighting large gaps that still exist in our understanding of the final stages of chromosome duplication and the evolutionary advantage of having a replication fork trap.

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What is all this fuss about Tus? Comparison of recent findings from biophysical and biochemical experiments

Cited by 16 publications

References 28 publications

Structure of the replication regulator Sap1 reveals functionally important interfaces

Structure of the replication regulator Sap1 reveals functionally important interfaces

Approaching Protein Barriers: Emerging Mechanisms of Replication Pausing in Eukaryotes

Termination of DNA replication at Tus-terbarriers results in under-replication of template DNA

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