Termination of DNA replication at Tus-ter barriers results in under-replication of template DNA

Mapping Intimacies ◽

10.1101/2021.02.25.432933 ◽

2021 ◽

Author(s):

Katie H. Jameson ◽

Christian J. Rudolph ◽

Michelle Hawkins

Keyword(s):

Dna Replication ◽

Replication Fork ◽

Genome Stability ◽

Processing Enzymes ◽

Replication Fork Progression ◽

Evolutionary Advantage ◽

Domains Of Life ◽

Dna Template ◽

Template Dna

ABSTRACTThe 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 15-24 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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Prokaryotic DNA replication mechanisms

Philosophical Transactions of the Royal Society of London Series B Biological Sciences ◽

10.1098/rstb.1987.0068 ◽

1987 ◽

Vol 317 (1187) ◽

pp. 395-420 ◽

Cited By ~ 57

Keyword(s):

Dna Replication ◽

Dna Polymerase ◽

Replication Fork ◽

Kinetic Studies ◽

Dna Helicase ◽

Gene 32 ◽

Dna Template ◽

Lagging Strand

The three different prokaryotic replication systems that have been most extensively studied use the same basic components for moving a DNA replication fork, even though the individual proteins are different and lack extensive amino acid sequence homology. In the T4 bacteriophage system, the components of the DNA replication complex can be grouped into functional classes as follows: DNA polymerase (gene 43 protein), helix-destabilizing protein (gene 32 protein), polymerase accessory proteins (gene 44/62 and 45 proteins), and primosome proteins (gene 41 DNA helicase and gene 61 RNA primase). DNA synthesis in the in vitro system starts by covalent addition onto the 3'OH end at a random nick on a double-stranded DNA template and proceeds to generate a replication fork that moves at about the in vivo rate, and with approximately the in vivo base-pairing fidelity. DNA is synthesized at the fork in a continuous fashion on the leading strand and in a discontinuous fashion on the lagging strand (generating short Okazaki fragments with 5'-linked pppApCpXpYpZ pentaribonucleotide primers). Kinetic studies reveal that the DNA polymerase molecule on the lagging strand stays associated with the fork as it moves. Therefore the DNA template on the lagging strand must be folded so that the stop site for the synthesis of one Okazaki fragment is adjacent to the start site for the next such fragment, allowing the polymerase and other replication proteins on the lagging strand to recycle.

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New histone supply regulates replication fork speed and PCNA unloading

The Journal of Cell Biology ◽

10.1083/jcb.201305017 ◽

2013 ◽

Vol 204 (1) ◽

pp. 29-43 ◽

Cited By ~ 89

Author(s):

Jakob Mejlvang ◽

Yunpeng Feng ◽

Constance Alabert ◽

Kai J. Neelsen ◽

Zuzana Jasencakova ◽

...

Keyword(s):

Dna Replication ◽

Replication Fork ◽

Nucleosome Occupancy ◽

Nucleosome Assembly ◽

Replication Fork Progression ◽

In Vivo Analysis ◽

Chromatin Integrity ◽

Canonical Histone

Correct duplication of DNA sequence and its organization into chromatin is central to genome function and stability. However, it remains unclear how cells coordinate DNA synthesis with provision of new histones for chromatin assembly to ensure chromosomal stability. In this paper, we show that replication fork speed is dependent on new histone supply and efficient nucleosome assembly. Inhibition of canonical histone biosynthesis impaired replication fork progression and reduced nucleosome occupancy on newly synthesized DNA. Replication forks initially remained stable without activation of conventional checkpoints, although prolonged histone deficiency generated DNA damage. PCNA accumulated on newly synthesized DNA in cells lacking new histones, possibly to maintain opportunity for CAF-1 recruitment and nucleosome assembly. Consistent with this, in vitro and in vivo analysis showed that PCNA unloading is delayed in the absence of nucleosome assembly. We propose that coupling of fork speed and PCNA unloading to nucleosome assembly provides a simple mechanism to adjust DNA replication and maintain chromatin integrity during transient histone shortage.

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Wss1 promotes replication stress tolerance by degrading histones

10.1101/2019.12.13.876110 ◽

2019 ◽

Author(s):

Karthik Maddi ◽

Daniel Kwesi Sam ◽

Florian Bonn ◽

Stefan Prgomet ◽

Eric Tulowetzke ◽

...

Keyword(s):

Dna Replication ◽

Protein Interactions ◽

Replication Fork ◽

Genome Stability ◽

Replication Stress ◽

Underlying Mechanism ◽

Adverse Conditions ◽

Replication Fork Progression ◽

Timely Completion ◽

Protein Crosslinks

SummaryTimely completion of DNA replication is central to accurate cell division and to the maintenance of genomic stability. However, certain DNA-protein interactions can physically impede DNA replication fork progression. Cells remove or bypass these physical impediments by different mechanisms to preserve DNA macromolecule integrity and genome stability. In Saccharomyces cerevisiae, Wss1, the DNA-protein crosslink repair protease, allows cells to tolerate hydroxyurea-induced replication stress but the underlying mechanism by which Wss1 promotes this function has remained unknown. Here we report that Wss1 provides cells tolerance to replication stress by directly degrading core histone subunits that non-specifically and non-covalently bind to single-stranded DNA. Unlike Wss1-dependent proteolysis of covalent DNA-protein crosslinks, proteolysis of histones does not require Cdc48 nor SUMO-binding activities. Wss1 thus acts as a multi-functional protease capable of targeting a broad range of covalent and non-covalent DNA-binding proteins to preserve genome stability during adverse conditions.

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Key histone chaperones have distinct roles in replisome progression and genomic stability

10.1101/2020.02.25.964221 ◽

2020 ◽

Author(s):

Ioannis Tsirkas ◽

Daniel Dovrat ◽

Yang Lei ◽

Angeliki Kalyva ◽

Diana Lotysh ◽

...

Keyword(s):

Dna Replication ◽

Chromatin Structure ◽

Replication Fork ◽

Genome Stability ◽

Spontaneous Mutation ◽

Genomic Stability ◽

Yeast Cells ◽

Eukaryotic Cells ◽

Histone Chaperones ◽

Replication Fork Progression

AbstractReplication-coupled (RC) nucleosome assembly is an essential process in eukaryotic cells in order to maintain chromatin structure during DNA replication. The deposition of newly synthesized H3/H4 histones during DNA replication is facilitated by specialized histone chaperones. Although the contribution of these histone chaperones to genomic stability has been thoroughly investigated, their effect on replisome progression is much less understood. By exploiting a time-lapse microscopy system for monitoring DNA replication in individual live cells, we examined how mutations in key histone chaperones including CAC1, RTT106, RTT109 and ASF1, affect replication fork progression. Our experiments revealed that mutations in CAC1 or RTT106 that directly deposit histones on the DNA, slowdown replication fork progression. In contrast, analysis of cells mutated in the intermediary ASF1 or RTT109 histone chaperones revealed that replisome progression is not affected. We found that mutations in histone chaperones including ASF1 and RTT109 lead to extended G2/M duration, elevated number of RPA foci and in some cases, increased spontaneous mutation rate. Our research suggests that histone chaperones have distinct roles in enabling high replisome progression and maintaining genome stability during cell cycle progression.Author SummaryHistone chaperones (HC) play key roles in maintaining the chromatin structure during DNA replication in eukaryotic cells. Despite extensive studies on HCs, little is known regarding their importance for replication fork progression during S-phase. Here, we utilized a live-cell imaging approach to measure the progression rates of single replication forks in individual yeast cells mutated in key histone chaperones. Using this approach, we show that mutations in CAC1 or RTT106 HCs that directly deposit histones on the DNA lead to slowdown of replication fork progression. In contrast, mutations in ASF1 or RTT109 HCs that transfers H3/H4 to CAC1 or RTT106, do not affect replisome progression but lead to post replication defects. Our results reveal distinct functions of HCs in replication fork progression and maintaining genome stability.

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Interferon-stimulated gene 15 accelerates replication fork progression inducing chromosomal breakage

The Journal of Cell Biology ◽

10.1083/jcb.202002175 ◽

2020 ◽

Vol 219 (8) ◽

Cited By ~ 5

Author(s):

Maria Chiara Raso ◽

Nikola Djoric ◽

Franziska Walser ◽

Sandra Hess ◽

Fabian Marc Schmid ◽

...

Keyword(s):

Dna Damage ◽

Dna Replication ◽

Replication Fork ◽

Genome Stability ◽

Dna Helicase ◽

Replication Forks ◽

Chromosomal Breakage ◽

Replication Fork Progression ◽

Ubiquitin System ◽

Interferon Stimulated Gene 15

DNA replication is highly regulated by the ubiquitin system, which plays key roles upon stress. The ubiquitin-like modifier ISG15 (interferon-stimulated gene 15) is induced by interferons, bacterial and viral infection, and DNA damage, but it is also constitutively expressed in many types of cancer, although its role in tumorigenesis is still largely elusive. Here, we show that ISG15 localizes at the replication forks, in complex with PCNA and the nascent DNA, where it regulates DNA synthesis. Indeed, high levels of ISG15, intrinsic or induced by interferon-β, accelerate DNA replication fork progression, resulting in extensive DNA damage and chromosomal aberrations. This effect is largely independent of ISG15 conjugation and relies on ISG15 functional interaction with the DNA helicase RECQ1, which promotes restart of stalled replication forks. Additionally, elevated ISG15 levels sensitize cells to cancer chemotherapeutic treatments. We propose that ISG15 up-regulation exposes cells to replication stress, impacting genome stability and response to genotoxic drugs.

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Base-pairing properties of O6-methylguanine in template DNA during in vitro DNA replication.

Journal of Biological Chemistry ◽

10.1016/s0021-9258(17)39698-9 ◽

1984 ◽

Vol 259 (13) ◽

pp. 8095-8100

Author(s):

E T Snow ◽

R S Foote ◽

S Mitra

Keyword(s):

Dna Replication ◽

Base Pairing ◽

Template Dna

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A concomitant loss of dormant origins and FANCC exacerbates genome instability by impairing DNA replication fork progression

Nucleic Acids Research ◽

10.1093/nar/gku170 ◽

2014 ◽

Vol 42 (9) ◽

pp. 5605-5615 ◽

Cited By ~ 17

Author(s):

Spencer W. Luebben ◽

Tsuyoshi Kawabata ◽

Charles S. Johnson ◽

M. Gerard O'Sullivan ◽

Naoko Shima

Keyword(s):

Dna Replication ◽

Replication Fork ◽

Genome Instability ◽

Replication Fork Progression

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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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Chromatin-associated degradation is defined by UBXN-3/FAF1 to safeguard DNA replication fork progression

Nature Communications ◽

10.1038/ncomms10612 ◽

2016 ◽

Vol 7 (1) ◽

Cited By ~ 15

Author(s):

André Franz ◽

Paul A. Pirson ◽

Domenic Pilger ◽

Swagata Halder ◽

Divya Achuthankutty ◽

...

Keyword(s):

Dna Replication ◽

Dynamic Process ◽

Metabolic Pathways ◽

Replication Fork ◽

Genome Instability ◽

Genome Integrity ◽

Substrate Selection ◽

Replication Fork Progression ◽

Replication Factors ◽

Spatiotemporal Control

Abstract The coordinated activity of DNA replication factors is a highly dynamic process that involves ubiquitin-dependent regulation. In this context, the ubiquitin-directed ATPase CDC-48/p97 recently emerged as a key regulator of chromatin-associated degradation in several of the DNA metabolic pathways that assure genome integrity. However, the spatiotemporal control of distinct CDC-48/p97 substrates in the chromatin environment remained unclear. Here, we report that progression of the DNA replication fork is coordinated by UBXN-3/FAF1. UBXN-3/FAF1 binds to the licensing factor CDT-1 and additional ubiquitylated proteins, thus promoting CDC-48/p97-dependent turnover and disassembly of DNA replication factor complexes. Consequently, inactivation of UBXN-3/FAF1 stabilizes CDT-1 and CDC-45/GINS on chromatin, causing severe defects in replication fork dynamics accompanied by pronounced replication stress and eventually resulting in genome instability. Our work identifies a critical substrate selection module of CDC-48/p97 required for chromatin-associated protein degradation in both Caenorhabditis elegans and humans, which is relevant to oncogenesis and aging.

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