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  1. Article ; Online: Regulation of T7 gp2.5 binding dynamics by its C-terminal tail, template conformation and sequence.

    Xu, Longfu / Cabanas-Danés, Jordi / Halma, Matthew T J / Heller, Iddo / Stratmann, Sarah A / van Oijen, Antoine M / Lee, Seung-Joo / Peterman, Erwin J G / Wuite, Gijs J L

    Nucleic acids research

    2023  Volume 51, Issue 13, Page(s) 6540–6553

    Abstract: Bacteriophage T7 single-stranded DNA-binding protein (gp2.5) binds to and protects transiently exposed regions of single-stranded DNA (ssDNA) while dynamically interacting with other proteins of the replication complex. We directly visualize ... ...

    Abstract Bacteriophage T7 single-stranded DNA-binding protein (gp2.5) binds to and protects transiently exposed regions of single-stranded DNA (ssDNA) while dynamically interacting with other proteins of the replication complex. We directly visualize fluorescently labelled T7 gp2.5 binding to ssDNA at the single-molecule level. Upon binding, T7 gp2.5 reduces the contour length of ssDNA by stacking nucleotides in a force-dependent manner, suggesting T7 gp2.5 suppresses the formation of secondary structure. Next, we investigate the binding dynamics of T7 gp2.5 and a deletion mutant lacking 21 C-terminal residues (gp2.5-Δ21C) under various template tensions. Our results show that the base sequence of the DNA molecule, ssDNA conformation induced by template tension, and the acidic terminal domain from T7 gp2.5 significantly impact on the DNA binding parameters of T7 gp2.5. Moreover, we uncover a unique template-catalyzed recycling behaviour of T7 gp2.5, resulting in an apparent cooperative binding to ssDNA, facilitating efficient spatial redistribution of T7 gp2.5 during the synthesis of successive Okazaki fragments. Overall, our findings reveal an efficient binding mechanism that prevents the formation of secondary structures by enabling T7 gp2.5 to rapidly rebind to nearby exposed ssDNA regions, during lagging strand DNA synthesis.
    MeSH term(s) Bacteriophage T7/genetics ; DNA/metabolism ; DNA Replication ; DNA, Single-Stranded/genetics ; DNA, Single-Stranded/metabolism ; Molecular Conformation ; Viral Proteins/metabolism
    Chemical Substances DNA (9007-49-2) ; DNA, Single-Stranded ; Viral Proteins
    Language English
    Publishing date 2023-05-30
    Publishing country England
    Document type Journal Article ; Research Support, Non-U.S. Gov't
    ZDB-ID 186809-3
    ISSN 1362-4962 ; 1362-4954 ; 0301-5610 ; 0305-1048
    ISSN (online) 1362-4962 ; 1362-4954
    ISSN 0301-5610 ; 0305-1048
    DOI 10.1093/nar/gkad485
    Database MEDical Literature Analysis and Retrieval System OnLINE

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    In stock of ZB MED Cologne/Königswinter

    Zs.A 1067: Show issues Location:
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    Jg. 1995 - 2021: Lesesall (1.OG)
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  2. Article ; Online: DnaB helicase dynamics in bacterial DNA replication resolved by single-molecule studies.

    Spinks, Richard R / Spenkelink, Lisanne M / Stratmann, Sarah A / Xu, Zhi-Qiang / Stamford, N Patrick J / Brown, Susan E / Dixon, Nicholas E / Jergic, Slobodan / van Oijen, Antoine M

    Nucleic acids research

    2021  Volume 49, Issue 12, Page(s) 6804–6816

    Abstract: In Escherichia coli, the DnaB helicase forms the basis for the assembly of the DNA replication complex. The stability of DnaB at the replication fork is likely important for successful replication initiation and progression. Single-molecule experiments ... ...

    Abstract In Escherichia coli, the DnaB helicase forms the basis for the assembly of the DNA replication complex. The stability of DnaB at the replication fork is likely important for successful replication initiation and progression. Single-molecule experiments have significantly changed the classical model of highly stable replication machines by showing that components exchange with free molecules from the environment. However, due to technical limitations, accurate assessments of DnaB stability in the context of replication are lacking. Using in vitro fluorescence single-molecule imaging, we visualise DnaB loaded on forked DNA templates. That these helicases are highly stable at replication forks, indicated by their observed dwell time of ∼30 min. Addition of the remaining replication factors results in a single DnaB helicase integrated as part of an active replisome. In contrast to the dynamic behaviour of other replisome components, DnaB is maintained within the replisome for the entirety of the replication process. Interestingly, we observe a transient interaction of additional helicases with the replication fork. This interaction is dependent on the τ subunit of the clamp-loader complex. Collectively, our single-molecule observations solidify the role of the DnaB helicase as the stable anchor of the replisome, but also reveal its capacity for dynamic interactions.
    MeSH term(s) DNA Replication ; DNA-Directed DNA Polymerase ; DnaB Helicases/metabolism ; Escherichia coli/genetics ; Multienzyme Complexes ; Single Molecule Imaging
    Chemical Substances Multienzyme Complexes ; DNA synthesome (EC 2.7.7.-) ; DNA-Directed DNA Polymerase (EC 2.7.7.7) ; dnaB protein, E coli (EC 3.6.1.-) ; DnaB Helicases (EC 3.6.4.12)
    Language English
    Publishing date 2021-06-17
    Publishing country England
    Document type Journal Article ; Research Support, Non-U.S. Gov't
    ZDB-ID 186809-3
    ISSN 1362-4962 ; 1362-4954 ; 0301-5610 ; 0305-1048
    ISSN (online) 1362-4962 ; 1362-4954
    ISSN 0301-5610 ; 0305-1048
    DOI 10.1093/nar/gkab493
    Database MEDical Literature Analysis and Retrieval System OnLINE

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    In stock of ZB MED Cologne/Königswinter

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    Je nach Verfügbarkeit (siehe Angabe bei Bestand)
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    Jg. 1995 - 2021: Lesesall (1.OG)
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  3. Article ; Online: The innate immune sensor IFI16 recognizes foreign DNA in the nucleus by scanning along the duplex.

    Stratmann, Sarah A / Morrone, Seamus R / van Oijen, Antoine M / Sohn, Jungsan

    eLife

    2015  Volume 4, Page(s) e11721

    Abstract: The ability to recognize foreign double-stranded (ds)DNA of pathogenic origin in the intracellular environment is an essential defense mechanism of the human innate immune system. However, the molecular mechanisms underlying distinction between foreign ... ...

    Abstract The ability to recognize foreign double-stranded (ds)DNA of pathogenic origin in the intracellular environment is an essential defense mechanism of the human innate immune system. However, the molecular mechanisms underlying distinction between foreign DNA and host genomic material inside the nucleus are not understood. By combining biochemical assays and single-molecule techniques, we show that the nuclear innate immune sensor IFI16 one-dimensionally tracks long stretches of exposed foreign dsDNA to assemble into supramolecular signaling platforms. We also demonstrate that nucleosomes represent barriers that prevent IFI16 from targeting host DNA by directly interfering with these one-dimensional movements. This unique scanning-assisted assembly mechanism allows IFI16 to distinguish friend from foe and assemble into oligomers efficiently and selectively on foreign DNA.
    MeSH term(s) Cell Nucleus/metabolism ; DNA/immunology ; DNA/metabolism ; Humans ; Nuclear Proteins/metabolism ; Nucleosomes/metabolism ; Phosphoproteins/metabolism
    Chemical Substances Nuclear Proteins ; Nucleosomes ; Phosphoproteins ; IFI16 protein, human (148998-64-5) ; DNA (9007-49-2)
    Language English
    Publishing date 2015-12-16
    Publishing country England
    Document type Journal Article ; Research Support, Non-U.S. Gov't
    ZDB-ID 2687154-3
    ISSN 2050-084X ; 2050-084X
    ISSN (online) 2050-084X
    ISSN 2050-084X
    DOI 10.7554/eLife.11721
    Database MEDical Literature Analysis and Retrieval System OnLINE

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  4. Article ; Online: Simultaneous Real-Time Imaging of Leading and Lagging Strand Synthesis Reveals the Coordination Dynamics of Single Replisomes.

    Duderstadt, Karl E / Geertsema, Hylkje J / Stratmann, Sarah A / Punter, Christiaan M / Kulczyk, Arkadiusz W / Richardson, Charles C / van Oijen, Antoine M

    Molecular cell

    2016  Volume 64, Issue 6, Page(s) 1035–1047

    Abstract: The molecular machinery responsible for DNA replication, the replisome, must efficiently coordinate DNA unwinding with priming and synthesis to complete duplication of both strands. Due to the anti-parallel nature of DNA, the leading strand is copied ... ...

    Abstract The molecular machinery responsible for DNA replication, the replisome, must efficiently coordinate DNA unwinding with priming and synthesis to complete duplication of both strands. Due to the anti-parallel nature of DNA, the leading strand is copied continuously, while the lagging strand is produced by repeated cycles of priming, DNA looping, and Okazaki-fragment synthesis. Here, we report a multidimensional single-molecule approach to visualize this coordination in the bacteriophage T7 replisome by simultaneously monitoring the kinetics of loop growth and leading-strand synthesis. We show that loops in the lagging strand predominantly occur during priming and only infrequently support subsequent Okazaki-fragment synthesis. Fluorescence imaging reveals polymerases remaining bound to the lagging strand behind the replication fork, consistent with Okazaki-fragment synthesis behind and independent of the replication complex. Individual replisomes display both looping and pausing during priming, reconciling divergent models for the regulation of primer synthesis and revealing an underlying plasticity in replisome operation.
    MeSH term(s) Bacteriophage T7/genetics ; Bacteriophage T7/metabolism ; Bacteriophage T7/ultrastructure ; DNA/biosynthesis ; DNA/genetics ; DNA Primase/genetics ; DNA Primase/metabolism ; DNA Primase/ultrastructure ; DNA Replication ; DNA, Viral/genetics ; DNA, Viral/metabolism ; DNA, Viral/ultrastructure ; Kinetics ; Single Molecule Imaging/methods ; Time-Lapse Imaging/methods
    Chemical Substances DNA, Viral ; Okazaki fragments ; DNA (9007-49-2) ; DNA Primase (EC 2.7.7.-)
    Language English
    Publishing date 2016-11-23
    Publishing country United States
    Document type Journal Article ; Research Support, Non-U.S. Gov't
    ZDB-ID 1415236-8
    ISSN 1097-4164 ; 1097-2765
    ISSN (online) 1097-4164
    ISSN 1097-2765
    DOI 10.1016/j.molcel.2016.10.028
    Database MEDical Literature Analysis and Retrieval System OnLINE

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    In stock of ZB MED Cologne/Königswinter

    Zs.A 5055: Show issues Location:
    Je nach Verfügbarkeit (siehe Angabe bei Bestand)
    bis Jg. 1994: Bestellungen von Artikeln über das Online-Bestellformular
    Jg. 1995 - 2021: Lesesall (2.OG)
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  5. Article ; Online: Bisecting Microfluidic Channels with Metallic Nanowires Fabricated by Nanoskiving.

    Kalkman, Gerard A / Zhang, Yanxi / Monachino, Enrico / Mathwig, Klaus / Kamminga, Machteld E / Pourhossein, Parisa / Oomen, Pieter E / Stratmann, Sarah A / Zhao, Zhiyuan / van Oijen, Antoine M / Verpoorte, Elisabeth / Chiechi, Ryan C

    ACS nano

    2016  Volume 10, Issue 2, Page(s) 2852–2859

    Abstract: This paper describes the fabrication of millimeter-long gold nanowires that bisect the center of microfluidic channels. We fabricated the nanowires by nanoskiving and then suspended them over a trench in a glass structure. The channel was sealed by ... ...

    Abstract This paper describes the fabrication of millimeter-long gold nanowires that bisect the center of microfluidic channels. We fabricated the nanowires by nanoskiving and then suspended them over a trench in a glass structure. The channel was sealed by bonding it to a complementary poly(dimethylsiloxane) structure. The resulting structures place the nanowires in the region of highest flow, as opposed to the walls, where it approaches zero, and expose their entire surface area to fluid. We demonstrate active functionality, by constructing a hot-wire anemometer to measure flow through determining the change in resistance of the nanowire as a function of heat dissipation at low voltage (<5 V). Further, passive functionality is demonstrated by visualizing individual, fluorescently labeled DNA molecules attached to the wires. We measure rates of flow and show that, compared to surface-bound DNA strands, elongation saturates at lower rates of flow and background fluorescence from nonspecific binding is reduced.
    Language English
    Publishing date 2016-02-23
    Publishing country United States
    Document type Journal Article ; Research Support, Non-U.S. Gov't
    ISSN 1936-086X
    ISSN (online) 1936-086X
    DOI 10.1021/acsnano.5b07996
    Database MEDical Literature Analysis and Retrieval System OnLINE

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