DNA molecular combing-based replication fork directionality profiling

AbstractThe replication strategy of metazoan genomes is still unclear, mainly because definitive maps of replication origins are missing. High-throughput methods are based on population average and thus may exclusively identify efficient initiation sites, whereas inefficient origins go undetected. Single-molecule analyses of specific loci can detect both common and rare initiation events along the targeted regions. However, these usually concentrate on positioning individual events, which only gives an overview of the replication dynamics. Here, we computed the replication fork directionality (RFD) profiles of two large genes in different transcriptional states in chicken DT40 cells, namely untranscribed and transcribed DMD and CCSER1 expressed at WT levels or overexpressed, by aggregating hundreds of oriented replication tracks detected on individual DNA fibres stretched by molecular combing. These profiles reconstituted RFD domains composed of zones of initiation flanking a zone of termination originally observed in mammalian genomes and were highly consistent with independent population-averaging profiles generated by Okazaki fragment sequencing. Importantly, we demonstrate that inefficient origins do not appear as detectable RFD shifts, explaining why dispersed initiation has remained invisible to population-based assays. Our method can both generate quantitative profiles and identify discrete events, thereby constituting a comprehensive approach to study metazoan genome replication.

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

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.1163864.628373 ◽

2009 ◽

Author(s):

Nick Rhind

Keyword(s):

Single Molecule ◽

Replication Fork ◽

Replication Fork Progression ◽

Single Molecule Analysis ◽

Lagging Strand

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

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.1163864.625689 ◽

2009 ◽

Author(s):

William Holloman

Keyword(s):

Single Molecule ◽

Replication Fork ◽

Replication Fork Progression ◽

Single Molecule Analysis ◽

Lagging Strand

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Developmental differences in genome replication program and origin activation

Nucleic Acids Research ◽

10.1093/nar/gkaa1124 ◽

2020 ◽

Vol 48 (22) ◽

pp. 12751-12777

Author(s):

Cathia Rausch ◽

Patrick Weber ◽

Paulina Prorok ◽

David Hörl ◽

Andreas Maiser ◽

...

Keyword(s):

Dna Replication ◽

Single Molecule ◽

Embryonic Stem ◽

S Phase ◽

Developmental Differences ◽

Centromeric Heterochromatin ◽

Genome Replication ◽

Origin Activation ◽

Spatio Temporal ◽

Mes Cells

Abstract To ensure error-free duplication of all (epi)genetic information once per cell cycle, DNA replication follows a cell type and developmental stage specific spatio-temporal program. Here, we analyze the spatio-temporal DNA replication progression in (un)differentiated mouse embryonic stem (mES) cells. Whereas telomeres replicate throughout S-phase, we observe mid S-phase replication of (peri)centromeric heterochromatin in mES cells, which switches to late S-phase replication upon differentiation. This replication timing reversal correlates with and depends on an increase in condensation and a decrease in acetylation of chromatin. We further find synchronous duplication of the Y chromosome, marking the end of S-phase, irrespectively of the pluripotency state. Using a combination of single-molecule and super-resolution microscopy, we measure molecular properties of the mES cell replicon, the number of replication foci active in parallel and their spatial clustering. We conclude that each replication nanofocus in mES cells corresponds to an individual replicon, with up to one quarter representing unidirectional forks. Furthermore, with molecular combing and genome-wide origin mapping analyses, we find that mES cells activate twice as many origins spaced at half the distance than somatic cells. Altogether, our results highlight fundamental developmental differences on progression of genome replication and origin activation in pluripotent cells.

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Alcohol consumption and cardiorespiratory fitness in five population-based studies

European Journal of Preventive Cardiology ◽

10.1177/2047487317738594 ◽

2017 ◽

Vol 25 (2) ◽

pp. 164-172 ◽

Cited By ~ 9

Author(s):

Sebastian E Baumeister ◽

Jonas D Finger ◽

Sven Gläser ◽

Marcus Dörr ◽

Marcello RP Markus ◽

...

Keyword(s):

Alcohol Consumption ◽

Oxygen Uptake ◽

Cardiorespiratory Fitness ◽

Polynomial Regression ◽

Population Based ◽

Peak Oxygen Uptake ◽

Heavy Episodic Drinking ◽

P Value ◽

Independent Population ◽

Average Alcohol

Background Poor cardiorespiratory fitness is a risk factor for cardiovascular morbidity. Alcohol consumption contributes substantially to the burden of disease, but its association with cardiorespiratory fitness is not well described. We examined associations between average alcohol consumption, heavy episodic drinking and cardiorespiratory fitness. Design The design of this study was as a cross-sectional population-based random sample. Methods We analysed data from five independent population-based studies (Study of Health in Pomerania (2008–2012); German Health Interview and Examination Survey (2008–2011); US National Health and Nutrition Examination Survey (NHANES) 1999–2000; NHANES 2001–2002; NHANES 2003–2004) including 7358 men and women aged 20–85 years, free of lung disease or asthma. Cardiorespiratory fitness, quantified by peak oxygen uptake, was assessed using exercise testing. Information regarding average alcohol consumption (ethanol in grams per day (g/d)) and heavy episodic drinking (5+ or 6+ drinks/occasion) was obtained from self-reports. Fractional polynomial regression models were used to determine the best-fitting dose-response relationship. Results Average alcohol consumption displayed an inverted U-type relation with peak oxygen uptake ( p-value<0.0001), after adjustment for age, sex, education, smoking and physical activity. Compared to individuals consuming 10 g/d (moderate consumption), current abstainers and individuals consuming 50 and 60 g/d had significantly lower peak oxygen uptake values (ml/kg/min) (β coefficients = −1.90, β = −0.06, β = −0.31, respectively). Heavy episodic drinking was not associated with peak oxygen uptake. Conclusions Across multiple adult population-based samples, moderate drinkers displayed better fitness than current abstainers and individuals with higher average alcohol consumption.

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Single-molecule imaging of DNA gyrase activity in livingEscherichia coli

10.1101/460006 ◽

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.

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Soft drinks consumption, diet quality and BMI in a Mediterranean population

Public Health Nutrition ◽

10.1017/s1368980010002788 ◽

2010 ◽

Vol 14 (5) ◽

pp. 778-784 ◽

Cited By ~ 6

Author(s):

Eva Balcells ◽

Mario Delgado-Noguera ◽

Ricardo Pardo-Lozano ◽

Taïs Roig-González ◽

Anna Renom ◽

...

Keyword(s):

Mediterranean Diet ◽

Sedentary Lifestyle ◽

Population Based ◽

Soft Drinks ◽

Mediterranean Population ◽

Regression Analyses ◽

Cross Sectional ◽

Independent Population ◽

The Mean ◽

Diet Score

AbstractObjectivesEvidence of the effects of soft drinks consumption on BMI and lifestyle in adult populations is mixed and quite limited. The aim of the present study was to determine the association of soft drinks consumption with BMI and lifestyle in a representative Mediterranean population.DesignTwo independent, population-based, cross-sectional (2000 and 2005) studies. Dietary intake was assessed using a validated FFQ. Weight and height were measured.SettingGirona, Spain.SubjectsRandom sample of the 35- to 74-year-old population (3910 men and 4285 women).ResultsLess than half (41·7 %) of the population consumed soft drinks; the mean consumption was 36·2 ml/d. The prevalence of sedentary lifestyle increased with the frequency of soft drinks consumption (P = 0·025). Daily soft drinks consumption significantly increased the risk of low adherence to the Mediterranean diet (OR = 0·57, 95 % CI 0·44, 0·74 v. top tertile of Mediterranean diet score). Multiple linear regression analyses, controlled for potential confounders, revealed that an increment in soft drinks consumption of 100 ml was associated with a 0·21 kg/m2 increase in BMI (P = 0·001). Only implausibly low reports of energy consumption showed a null association between soft drinks consumption and BMI.ConclusionsSoft drinks consumption was not embedded in a healthy diet context and was positively associated with BMI and sedentary lifestyle in this Mediterranean population.

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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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CTCF and cohesin regulate chromatin loop stability with distinct dynamics

eLife ◽

10.7554/elife.25776 ◽

2017 ◽

Vol 6 ◽

Cited By ~ 208

Author(s):

Anders S Hansen ◽

Iryna Pustova ◽

Claudia Cattoglio ◽

Robert Tjian ◽

Xavier Darzacq

Keyword(s):

Cell Cycle ◽

Gene Regulation ◽

Single Molecule ◽

Stable Complex ◽

Search Process ◽

Chromatin Loop ◽

Domain Formation ◽

Loop Domain ◽

Spatial Domains ◽

Mammalian Genomes

Folding of mammalian genomes into spatial domains is critical for gene regulation. The insulator protein CTCF and cohesin control domain location by folding domains into loop structures, which are widely thought to be stable. Combining genomic and biochemical approaches we show that CTCF and cohesin co-occupy the same sites and physically interact as a biochemically stable complex. However, using single-molecule imaging we find that CTCF binds chromatin much more dynamically than cohesin (~1–2 min vs. ~22 min residence time). Moreover, after unbinding, CTCF quickly rebinds another cognate site unlike cohesin for which the search process is long (~1 min vs. ~33 min). Thus, CTCF and cohesin form a rapidly exchanging 'dynamic complex' rather than a typical stable complex. Since CTCF and cohesin are required for loop domain formation, our results suggest that chromatin loops are dynamic and frequently break and reform throughout the cell cycle.

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Interferon beta in relapsing-remitting multiple sclerosis: an independent postmarketing study in southern Italy

Multiple Sclerosis Journal ◽

10.1191/1352458503ms948oa ◽

2003 ◽

Vol 9 (5) ◽

pp. 451-457 ◽

Cited By ~ 52

Author(s):

Maria Trojano ◽

Maria Liguori ◽

Damiano Paolicelli ◽

Giovanni Bosco Zimatore ◽

Francesca De Robertis ◽

...

Keyword(s):

Multiple Sclerosis ◽

Adverse Events ◽

Interferon Beta ◽

Population Based ◽

First Year ◽

Independent Population ◽

Disability Status ◽

Relapsing Remitting ◽

Remitting Multiple Sclerosis ◽

Relapsing Remitting Multiple Sclerosis

This independent, population-based surveillance study monitored the efficacy and safety of interferon beta (IFNb) products in 1033 patients with relapsing -remitting multiple sclerosis (RRMS) from 15 centres in Italy. Relapses, Expanded Disability Status Scale (EDSS) scores, and adverse events were evaluated for up to 24 months. Data of patients with a baseline EDSS score 5-3.5 are reported. The proportions of relapse-free patients were similar among the groups at 12 and 24 months (P =0.10). IFNb products produced significant reductio ns from baseline in relapse rates at 12 and 24 months (P B-0.001), with no differences among treatments (P =0.2). There were no significant differences in mean EDSS change among groups at 12 or 24 months. The IFNb-1b group showed a higher incidence of adverse events during the first year of treatment (P B-0.05) than IFNb-1a groups, and more withdrawals (10%) compared with Avonex (5%) at 24 months. IFNb products are equally effective in low disability RRMS, but IFNb-1a may have a more favorable efficacy/tolerability ratio.

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