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  1. Article: E. coli

    Fazio, Nicole / Mersch, Kacey N / Hao, Linxuan / Lohman, Timothy M

    bioRxiv : the preprint server for biology

    2023  

    Abstract: Much is still unknown about the mechanisms by which helicases unwind duplex DNA. Whereas structure-based models describe DNA unwinding as a consequence of mechanically pulling the DNA duplex across a wedge domain in the helicase by the single stranded ( ... ...

    Abstract Much is still unknown about the mechanisms by which helicases unwind duplex DNA. Whereas structure-based models describe DNA unwinding as a consequence of mechanically pulling the DNA duplex across a wedge domain in the helicase by the single stranded (ss)DNA translocase activity of the ATPase motors, biochemical data indicate that processive DNA unwinding by the
    Language English
    Publishing date 2023-10-17
    Publishing country United States
    Document type Preprint
    DOI 10.1101/2023.10.13.561901
    Database MEDical Literature Analysis and Retrieval System OnLINE

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  2. Article ; Online: E. coli RecB Nuclease Domain Regulates RecBCD Helicase Activity but not Single Stranded DNA Translocase Activity.

    Fazio, Nicole T / Mersch, Kacey N / Hao, Linxuan / Lohman, Timothy M

    Journal of molecular biology

    2023  Volume 436, Issue 2, Page(s) 168381

    Abstract: Much is still unknown about the mechanisms by which helicases unwind duplex DNA. Whereas structure-based models describe DNA unwinding as occurring by the ATPase motors mechanically pulling the DNA duplex across a wedge domain in the helicase, ... ...

    Abstract Much is still unknown about the mechanisms by which helicases unwind duplex DNA. Whereas structure-based models describe DNA unwinding as occurring by the ATPase motors mechanically pulling the DNA duplex across a wedge domain in the helicase, biochemical data show that processive DNA unwinding by E. coli RecBCD helicase can occur in the absence of ssDNA translocation by the canonical RecB and RecD motors. Here we show that DNA unwinding is not a simple consequence of ssDNA translocation by the motors. Using stopped-flow fluorescence approaches, we show that a RecB nuclease domain deletion variant (RecB
    MeSH term(s) DNA Helicases/chemistry ; DNA Helicases/genetics ; DNA, Single-Stranded/chemistry ; Escherichia coli/genetics ; Escherichia coli/metabolism ; Escherichia coli Proteins/chemistry ; Escherichia coli Proteins/genetics ; Exodeoxyribonuclease V/chemistry ; Exodeoxyribonuclease V/genetics ; Protein Domains
    Chemical Substances DNA Helicases (EC 3.6.4.-) ; DNA, Single-Stranded ; Escherichia coli Proteins ; Exodeoxyribonuclease V (EC 3.1.11.5) ; exodeoxyribonuclease V, E coli (EC 3.1.11.5)
    Language English
    Publishing date 2023-12-09
    Publishing country Netherlands
    Document type Journal Article ; Research Support, N.I.H., Extramural
    ZDB-ID 80229-3
    ISSN 1089-8638 ; 0022-2836
    ISSN (online) 1089-8638
    ISSN 0022-2836
    DOI 10.1016/j.jmb.2023.168381
    Database MEDical Literature Analysis and Retrieval System OnLINE

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    In stock of ZB MED Cologne/Königswinter

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  3. Article ; Online: "Helicase" Activity promoted through dynamic interactions between a ssDNA translocase and a diffusing SSB protein.

    Mersch, Kacey N / Sokoloski, Joshua E / Nguyen, Binh / Galletto, Roberto / Lohman, Timothy M

    Proceedings of the National Academy of Sciences of the United States of America

    2023  Volume 120, Issue 15, Page(s) e2216777120

    Abstract: Replication protein A (RPA) is a eukaryotic single-stranded (ss) DNA-binding (SSB) protein that is essential for all aspects of genome maintenance. RPA binds ssDNA with high affinity but can also diffuse along ssDNA. By itself, RPA is capable of ... ...

    Abstract Replication protein A (RPA) is a eukaryotic single-stranded (ss) DNA-binding (SSB) protein that is essential for all aspects of genome maintenance. RPA binds ssDNA with high affinity but can also diffuse along ssDNA. By itself, RPA is capable of transiently disrupting short regions of duplex DNA by diffusing from a ssDNA that flanks the duplex DNA. Using single-molecule total internal reflection fluorescence and optical trapping combined with fluorescence approaches, we show that
    MeSH term(s) Humans ; Saccharomyces cerevisiae/genetics ; Saccharomyces cerevisiae/metabolism ; Protein Binding/genetics ; Replication Protein A/metabolism ; DNA, Single-Stranded/metabolism ; DNA/metabolism ; Adenosine Triphosphate/metabolism ; DNA Helicases/metabolism ; Saccharomyces cerevisiae Proteins/metabolism
    Chemical Substances Replication Protein A ; DNA, Single-Stranded ; DNA (9007-49-2) ; Adenosine Triphosphate (8L70Q75FXE) ; PIF1 protein, S cerevisiae (EC 3.6.1.-) ; DNA Helicases (EC 3.6.4.-) ; Saccharomyces cerevisiae Proteins
    Language English
    Publishing date 2023-04-03
    Publishing country United States
    Document type Journal Article ; Research Support, N.I.H., Extramural ; Research Support, Non-U.S. Gov't
    ZDB-ID 209104-5
    ISSN 1091-6490 ; 0027-8424
    ISSN (online) 1091-6490
    ISSN 0027-8424
    DOI 10.1073/pnas.2216777120
    Database MEDical Literature Analysis and Retrieval System OnLINE

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  4. Article: Altering CLC stoichiometry by reducing non-polar side-chains at the dimerization interface

    Mersch, Kacey / Ozturk, Tugba N / Park, Kunwoong / Lim, Hyun-Ho / Robertson, Janice L

    Elsevier Ltd Journal of molecular biology. 2021 Apr. 16, v. 433, no. 8

    2021  

    Abstract: CLC-ec1 is a Cl⁻/H⁺ antiporter that forms stable homodimers in lipid bilayers, with a free energy of −10.9 kcal/mol in 2:1 POPE/POPG lipid bilayers. The dimerization interface is formed by four transmembrane helices: H, I, P and Q, that are lined by non- ... ...

    Abstract CLC-ec1 is a Cl⁻/H⁺ antiporter that forms stable homodimers in lipid bilayers, with a free energy of −10.9 kcal/mol in 2:1 POPE/POPG lipid bilayers. The dimerization interface is formed by four transmembrane helices: H, I, P and Q, that are lined by non-polar side-chains that come in close contact, yet it is unclear as to whether their interactions drive dimerization. To investigate whether non-polar side-chains are required for dimer assembly, we designed a series of constructs where side-chain packing in the dimer state is significantly reduced by making 4–5 alanine substitutions along each helix (H-ala, I-ala, P-ala, Q-ala). All constructs are functional and three purify as stable dimers in detergent micelles despite the removal of significant side-chain interactions. On the other hand, H-ala shows the unique behavior of purifying as a mixture of monomers and dimers, followed by a rapid and complete conversion to monomers. In lipid bilayers, all four constructs are monomeric as examined by single-molecule photobleaching analysis. Further study of the H-helix shows that the single mutation L194A is sufficient to yield monomeric CLC-ec1 in detergent micelles and lipid bilayers. X-ray crystal structures of L194A reveal the protein re-assembles to form dimers, with a structure that is identical to wild-type. Altogether, these results demonstrate that non-polar membrane embedded side-chains play an important role in defining dimer stability, but the stoichiometry is highly contextual to the solvent environment. Furthermore, we discovered that L194 is a molecular hot-spot for defining dimerization of CLC-ec1.
    Keywords Gibbs free energy ; X-radiation ; alanine ; antiporters ; detergents ; dimerization ; lipid bilayers ; micelles ; molecular biology ; mutation ; photobleaching ; solvents ; stoichiometry
    Language English
    Dates of publication 2021-0416
    Publishing place Elsevier Ltd
    Document type Article
    Note NAL-AP-2-clean
    ZDB-ID 80229-3
    ISSN 1089-8638 ; 0022-2836
    ISSN (online) 1089-8638
    ISSN 0022-2836
    DOI 10.1016/j.jmb.2021.166886
    Database NAL-Catalogue (AGRICOLA)

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  5. Article ; Online: Altering CLC stoichiometry by reducing non-polar side-chains at the dimerization interface.

    Mersch, Kacey / Ozturk, Tugba N / Park, Kunwoong / Lim, Hyun-Ho / Robertson, Janice L

    Journal of molecular biology

    2021  Volume 433, Issue 8, Page(s) 166886

    Abstract: CLC-ec1 is a ... ...

    Abstract CLC-ec1 is a Cl
    MeSH term(s) Antiporters/chemistry ; Antiporters/genetics ; Dimerization ; Escherichia coli ; Escherichia coli Proteins/chemistry ; Escherichia coli Proteins/genetics ; Lipid Bilayers/chemistry ; Micelles ; Mutation ; Single Molecule Imaging
    Chemical Substances Antiporters ; CLC-ec1 protein, E coli ; Escherichia coli Proteins ; Lipid Bilayers ; Micelles
    Language English
    Publishing date 2021-02-20
    Publishing country Netherlands
    Document type Journal Article ; Research Support, N.I.H., Extramural ; Research Support, Non-U.S. Gov't
    ZDB-ID 80229-3
    ISSN 1089-8638 ; 0022-2836
    ISSN (online) 1089-8638
    ISSN 0022-2836
    DOI 10.1016/j.jmb.2021.166886
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    Zs.A 867: Show issues Location:
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  6. Article ; Online: Gβ1 is required for neutrophil migration in zebrafish.

    Ke, Wenfan / Ye, Ding / Mersch, Kacey / Xu, Hui / Chen, Songhai / Lin, Fang

    Developmental biology

    2017  Volume 428, Issue 1, Page(s) 135–147

    Abstract: Signaling mediated by G protein-coupled receptors (GPCRs) is essential for the migration of cells toward chemoattractants. The recruitment of neutrophils to injured tissues in zebrafish larvae is a useful model for studying neutrophil migration and ... ...

    Abstract Signaling mediated by G protein-coupled receptors (GPCRs) is essential for the migration of cells toward chemoattractants. The recruitment of neutrophils to injured tissues in zebrafish larvae is a useful model for studying neutrophil migration and trafficking in vivo. Indeed, the study of this process led to the discovery that PI3Kγ is required for the polarity and motility of neutrophils, features that are necessary for the directed migration of these cells to wounds. However, the mechanism by which PI3Kγ is activated remains to be determined. Here we show that signaling by specifically the heterotrimeric G protein subunit Gβ1 is critical for neutrophil migration in response to wounding. In embryos treated with small-molecule inhibitors of Gβγ signaling, neutrophils failed to migrate to wound sites. Although both the Gβ1 and Gβ4 isoforms are expressed in migrating neutrophils, only deficiency for the former (morpholino-based knockdown) interfered with the directed migration of neutrophils towards wounds. The Gβ1 deficiency also impaired the ability of cells to change cell shape and reduced their general motility, defects that are similar to those in neutrophils deficient for PI3Kγ. Transplantation assays showed that the requirement for Gβ1 in neutrophil migration is cell autonomous. Finally, live imaging revealed that Gβ1 is required for polarized activation of PI3K, and for the actin dynamics that enable neutrophil migration. Collectively, our data indicate that Gβ1 signaling controls proper neutrophil migration by activating PI3K and modulating actin dynamics. Moreover, they illustrate a role for a specific Gβ isoform in chemotaxis in vivo.
    MeSH term(s) Animals ; Animals, Genetically Modified ; Chemotaxis, Leukocyte/physiology ; Class Ib Phosphatidylinositol 3-Kinase/metabolism ; GTP-Binding Protein beta Subunits/antagonists & inhibitors ; GTP-Binding Protein beta Subunits/genetics ; GTP-Binding Protein beta Subunits/metabolism ; Heterotrimeric GTP-Binding Proteins/antagonists & inhibitors ; Heterotrimeric GTP-Binding Proteins/genetics ; Heterotrimeric GTP-Binding Proteins/metabolism ; Morpholinos/genetics ; Neutrophils/physiology ; Signal Transduction ; Wound Healing/physiology ; Zebrafish/embryology ; Zebrafish/metabolism ; Zebrafish Proteins/antagonists & inhibitors ; Zebrafish Proteins/genetics ; Zebrafish Proteins/metabolism
    Chemical Substances GTP-Binding Protein beta Subunits ; Morpholinos ; Zebrafish Proteins ; gnb1a protein, zebrafish ; Class Ib Phosphatidylinositol 3-Kinase (EC 2.7.1.137) ; Heterotrimeric GTP-Binding Proteins (EC 3.6.5.1)
    Language English
    Publishing date 2017-05-27
    Publishing country United States
    Document type Journal Article
    ZDB-ID 1114-9
    ISSN 1095-564X ; 0012-1606
    ISSN (online) 1095-564X
    ISSN 0012-1606
    DOI 10.1016/j.ydbio.2017.05.024
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    Zs.B 508: Show issues Location:
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  7. Article ; Online: The dimerization equilibrium of a ClC Cl(-)/H(+) antiporter in lipid bilayers.

    Chadda, Rahul / Krishnamani, Venkatramanan / Mersch, Kacey / Wong, Jason / Brimberry, Marley / Chadda, Ankita / Kolmakova-Partensky, Ludmila / Friedman, Larry J / Gelles, Jeff / Robertson, Janice L

    eLife

    2016  Volume 5

    Abstract: Interactions between membrane protein interfaces in lipid bilayers play an important role in membrane protein folding but quantification of the strength of these interactions has been challenging. Studying dimerization of ClC-type transporters offers a ... ...

    Abstract Interactions between membrane protein interfaces in lipid bilayers play an important role in membrane protein folding but quantification of the strength of these interactions has been challenging. Studying dimerization of ClC-type transporters offers a new approach to the problem, as individual subunits adopt a stable and functionally verifiable fold that constrains the system to two states - monomer or dimer. Here, we use single-molecule photobleaching analysis to measure the probability of ClC-ec1 subunit capture into liposomes during extrusion of large, multilamellar membranes. The capture statistics describe a monomer to dimer transition that is dependent on the subunit/lipid mole fraction density and follows an equilibrium dimerization isotherm. This allows for the measurement of the free energy of ClC-ec1 dimerization in lipid bilayers, revealing that it is one of the strongest membrane protein complexes measured so far, and introduces it as new type of dimerization model to investigate the physical forces that drive membrane protein association in membranes.
    MeSH term(s) Chloride Channels/metabolism ; Lipid Bilayers ; Protein Multimerization ; Single Molecule Imaging
    Chemical Substances CLC-1 channel ; Chloride Channels ; Lipid Bilayers
    Language English
    Publishing date 2016-08-03
    Publishing country England
    Document type Journal Article ; Research Support, Non-U.S. Gov't ; Research Support, N.I.H., Extramural
    ZDB-ID 2687154-3
    ISSN 2050-084X ; 2050-084X
    ISSN (online) 2050-084X
    ISSN 2050-084X
    DOI 10.7554/eLife.17438
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