scholarly journals Single-molecule analysis of DNA replication reveals novel features in the divergent eukaryotes Leishmania and Trypanosoma brucei versus mammalian cells

2016 ◽  
Vol 6 (1) ◽  
Author(s):  
Slavica Stanojcic ◽  
Lauriane Sollelis ◽  
Nada Kuk ◽  
Lucien Crobu ◽  
Yves Balard ◽  
...  
Keyword(s):  
Dna Replication ◽  
Trypanosoma Brucei ◽  
Single Molecule ◽  
Mammalian Cells ◽  
Single Molecule Analysis

scholarly journals Single molecule analysis of Trypanosoma brucei DNA replication dynamics

2015 ◽  
Vol 43 (5) ◽  
pp. 2655-2665 ◽  
Author(s):  
Simone Guedes Calderano ◽  
William C. Drosopoulos ◽  
Marina Mônaco Quaresma ◽  
Catarina A. Marques ◽  
Settapong Kosiyatrakul ◽  
...  
Keyword(s):  
Dna Replication ◽  
Trypanosoma Brucei ◽  
Single Molecule ◽  
Single Molecule Analysis

scholarly journals Beginning at the end: DNA replication within the telomere

2015 ◽  
Vol 210 (2) ◽  
pp. 177-179 ◽  
Author(s):  
Susan A. Gerbi
Keyword(s):  
Dna Replication ◽  
Mouse Chromosome ◽  
Single Molecule ◽  
Template Strand ◽  
Cell Biol ◽  
Chromosome Arm ◽  
Leading Strand ◽  
Wrn Helicase ◽  
Single Molecule Analysis

Using single molecule analysis of replicated DNA (SMARD), Drosopoulos et al. (2015; J. Cell Biol. http://dx.doi.org/10.1083/jcb.201410061) report that DNA replication initiates at measurable frequency within the telomere of mouse chromosome arm 14q. They demonstrate that resolution of G4 structures on the G-rich template strand of the telomere requires some overlapping functions of BLM and WRN helicase for leading strand synthesis.

scholarly journals Single-molecule analysis reveals self assembly and nanoscale segregation of two distinct cavin subcomplexes on caveolae

eLife ◽  
2014 ◽  
Vol 3 ◽  
Author(s):  
Yann Gambin ◽  
Nicholas Ariotti ◽  
Kerrie-Ann McMahon ◽  
Michele Bastiani ◽  
Emma Sierecki ◽  
...  
Keyword(s):  
Single Molecule ◽  
Self Assembly ◽  
Mammalian Cells ◽  
Protein Complexes ◽  
Building Blocks ◽  
Scaffolding Protein ◽  
Cell Extracts ◽  
Single Molecule Analysis ◽  
Exquisite Specificity

In mammalian cells three closely related cavin proteins cooperate with the scaffolding protein caveolin to form membrane invaginations known as caveolae. Here we have developed a novel single-molecule fluorescence approach to directly observe interactions and stoichiometries in protein complexes from cell extracts and from in vitro synthesized components. We show that up to 50 cavins associate on a caveola. However, rather than forming a single coat complex containing the three cavin family members, single-molecule analysis reveals an exquisite specificity of interactions between cavin1, cavin2 and cavin3. Changes in membrane tension can flatten the caveolae, causing the release of the cavin coat and its disassembly into separate cavin1-cavin2 and cavin1-cavin3 subcomplexes. Each of these subcomplexes contain 9 ± 2 cavin molecules and appear to be the building blocks of the caveolar coat. High resolution immunoelectron microscopy suggests a remarkable nanoscale organization of these separate subcomplexes, forming individual striations on the surface of caveolae.

Single molecule analysis of DNA replication

Biochimie ◽  
1999 ◽  
Vol 81 (8-9) ◽  
pp. 859-871 ◽  
Author(s):  
John Herrick ◽  
Aaron Bensimon
Keyword(s):  
Dna Replication ◽  
Single Molecule ◽  
Single Molecule Analysis

scholarly journals FANCM, BRCA1, and BLM cooperatively resolve the replication stress at the ALT telomeres

2017 ◽  
Vol 114 (29) ◽  
pp. E5940-E5949 ◽  
Author(s):  
Xiaolei Pan ◽  
William C. Drosopoulos ◽  
Louisa Sethi ◽  
Advaitha Madireddy ◽  
Carl L. Schildkraut ◽  
...  
Keyword(s):  
Single Molecule ◽  
Mammalian Cells ◽  
Synthetic Lethality ◽  
Replication Stress ◽  
Fragile Sites ◽  
Telomere Maintenance ◽  
Synthetic Lethal ◽  
Synthetic Lethal Interactions ◽  
Dna End Resection ◽  
Single Molecule Analysis

In the mammalian genome, certain genomic loci/regions pose greater challenges to the DNA replication machinery (i.e., the replisome) than others. Such known genomic loci/regions include centromeres, common fragile sites, subtelomeres, and telomeres. However, the detailed mechanism of how mammalian cells cope with the replication stress at these loci/regions is largely unknown. Here we show that depletion of FANCM, or of one of its obligatory binding partners, FAAP24, MHF1, and MHF2, induces replication stress primarily at the telomeres of cells that use the alternative lengthening of telomeres (ALT) pathway as their telomere maintenance mechanism. Using the telomere-specific single-molecule analysis of replicated DNA technique, we found that depletion of FANCM dramatically reduces the replication efficiency at ALT telomeres. We further show that FANCM, BRCA1, and BLM are actively recruited to the ALT telomeres that are experiencing replication stress and that the recruitment of BRCA1 and BLM to these damaged telomeres is interdependent and is regulated by both ATR and Chk1. Mechanistically, we demonstrated that, in FANCM-depleted ALT cells, BRCA1 and BLM help to resolve the telomeric replication stress by stimulating DNA end resection and homologous recombination (HR). Consistent with their roles in resolving the replication stress induced by FANCM deficiency, simultaneous depletion of BLM and FANCM, or of BRCA1 and FANCM, leads to increased micronuclei formation and synthetic lethality in ALT cells. We propose that these synthetic lethal interactions can be explored for targeting the ALT cancers.

scholarly journals Single-molecule analysis of DNA replication in Xenopus egg extracts

Methods ◽  
2012 ◽  
Vol 57 (2) ◽  
pp. 179-186 ◽  
Author(s):  
Hasan Yardimci ◽  
Anna B. Loveland ◽  
Antoine M. van Oijen ◽  
Johannes C. Walter
Keyword(s):  
Dna Replication ◽  
Single Molecule ◽  
Egg Extracts ◽  
Xenopus Egg ◽  
Xenopus Egg Extracts ◽  
Single Molecule Analysis

scholarly journals Single-molecule Analysis of DNA Replication Dynamics in Budding Yeast and Human Cells by DNA Combing

BIO-PROTOCOL ◽  
2017 ◽  
Vol 7 (11) ◽  
Author(s):  
Hélène Tourrière ◽  
Julie Saksouk ◽  
Armelle Lengronne ◽  
Philippe Pasero
Keyword(s):  
Dna Replication ◽  
Single Molecule ◽  
Budding Yeast ◽  
Human Cells ◽  
Dna Combing ◽  
Single Molecule Analysis

scholarly journals Sequential steps in DNA replication are inhibited to ensure reduction of ploidy in meiosis

2013 ◽  
Vol 24 (5) ◽  
pp. 578-587 ◽  
Author(s):  
Hui Hua ◽  
Mandana Namdar ◽  
Olivier Ganier ◽  
Juraj Gregan ◽  
Marcel Méchali ◽  
...  
Keyword(s):  
Dna Replication ◽  
Chromosome Segregation ◽  
Single Molecule ◽  
Replication Forks ◽  
Cyclin Dependent Kinase ◽  
Chromatin Binding ◽  
Control Factors ◽  
Spindle Disassembly ◽  
Single Molecule Analysis ◽  
Meiosis I

Meiosis involves two successive rounds of chromosome segregation without an intervening S phase. Exit from meiosis I is distinct from mitotic exit, in that replication origins are not licensed by Mcm2-7 chromatin binding, but spindle disassembly occurs during a transient interphase-like state before meiosis II. The absence of licensing is assumed to explain the block to DNA replication, but this has not been formally tested. Here we attempt to subvert this block by expressing the licensing control factors Cdc18 and Cdt1 during the interval between meiotic nuclear divisions. Surprisingly, this leads only to a partial round of DNA replication, even when these factors are overexpressed and effect clear Mcm2-7 chromatin binding. Combining Cdc18 and Cdt1 expression with modulation of cyclin-dependent kinase activity, activation of Dbf4-dependent kinase, or deletion of the Spd1 inhibitor of ribonucleotide reductase has little additional effect on the extent of DNA replication. Single-molecule analysis indicates this partial round of replication results from inefficient progression of replication forks, and thus both initiation and elongation replication steps may be inhibited in late meiosis. In addition, DNA replication or damage during the meiosis I–II interval fails to arrest meiotic progress, suggesting absence of checkpoint regulation of meiosis II entry.

scholarly journals Mapping DNA replication with nanopore sequencing

2018 ◽  
Author(s):  
Magali Hennion ◽  
Jean-Michel Arbona ◽  
Corinne Cruaud ◽  
Florence Proux ◽  
Benoît Le Tallec ◽  
...  
Keyword(s):  
Dna Replication ◽  
Single Molecule ◽  
Electrical Signal ◽  
Yeast Cells ◽  
Nanopore Sequencing ◽  
Single Molecule Level ◽  
Experimental Systems ◽  
Genome Wide ◽  
Single Molecule Analysis

ABSTRACTWe have harnessed nanopore sequencing to study DNA replication genome-wide at the single-molecule level. Using in vitro prepared DNA substrates, we characterized the effect of bromodeoxyuridine (BrdU) substitution for thymidine on the MinION nanopore electrical signal. Using a neural-network basecaller trained on yeast DNA containing either BrdU or thymidine, we identified BrdU-labelled tracts in yeast cells synchronously entering S phase in the presence of hydroxyurea and BrdU. As expected, the BrdU-labelled tracts coincided with previously identified early-firing, but not late-firing, replication origins. These results open the way to high-throughput, high-resolution, single-molecule analysis of DNA replication in many experimental systems.

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