scholarly journals ParB dynamics and the critical role of the CTD in DNA condensation unveiled by combined force-fluorescence measurements

2018 ◽  
Author(s):  
Julene Madariaga-Marcos ◽  
Cesar L. Pastrana ◽  
Gemma L. M. Fisher ◽  
Mark S. Dillingham ◽  
Fernando Moreno-Herrero
Keyword(s):  
Dna Binding ◽  
Protein Binding ◽  
Network Formation ◽  
Critical Role ◽  
Magnetic Tweezers ◽  
Dna Condensation ◽  
Full Length ◽  
Tirf Microscopy ◽  
Fluorescence Measurements

AbstractBacillus subtilis ParB forms multimeric networks involving non-specific DNA binding leading to DNA condensation. In our previous work (Fisher et al., 2017), we found that an excess of the free C-terminal domain (CTD) of ParB impeded DNA condensation or promoted decondensation of pre-assembled networks. However, interpretation of the molecular basis for this phenomenon was complicated by our inability to uncouple protein binding from DNA condensation. Here, we have combined lateral magnetic tweezers with TIRF microscopy to simultaneously control the restrictive force against condensation and to visualize ParB protein binding by fluorescence. At non-permissive forces for condensation, ParB binds non-specifically and highly dynamically to DNA. Our new approach concluded that the free CTD blocks the formation of ParB networks by heterodimerization with full length DNA-bound ParB. This strongly supports a model in which the CTD acts as a key bridging interface between distal DNA binding loci within ParB networks.Significance StatementUsing combined Magnetic Tweezers and TIRF microscopy we show that the CTD of ParB blocks ParB network formation by heterodimerization with the full-length protein, which remains bound to the DNA.

scholarly journals ParB dynamics and the critical role of the CTD in DNA condensation unveiled by combined force-fluorescence measurements

eLife ◽  
2019 ◽  
Vol 8 ◽  
Author(s):  
Julene Madariaga-Marcos ◽  
Cesar L Pastrana ◽  
Gemma LM Fisher ◽  
Mark Simon Dillingham ◽  
Fernando Moreno-Herrero
Keyword(s):  
Dna Binding ◽  
Protein Binding ◽  
Molecular Basis ◽  
Critical Role ◽  
Magnetic Tweezers ◽  
Dna Condensation ◽  
Uncouple Protein ◽  
New Approach ◽  
Fluorescence Measurements

Bacillus subtilis ParB forms multimeric networks involving non-specific DNA binding leading to DNA condensation. Previously, we found that an excess of the free C-terminal domain (CTD) of ParB impeded DNA condensation or promoted decondensation of pre-assembled networks (Fisher et al., 2017). However, interpretation of the molecular basis for this phenomenon was complicated by our inability to uncouple protein binding from DNA condensation. Here, we have combined lateral magnetic tweezers with TIRF microscopy to simultaneously control the restrictive force against condensation and to visualise ParB protein binding by fluorescence. At non-permissive forces for condensation, ParB binds non-specifically and highly dynamically to DNA. Our new approach concluded that the free CTD blocks the formation of ParB networks by heterodimerisation with full length DNA-bound ParB. This strongly supports a model in which the CTD acts as a key bridging interface between distal DNA binding loci within ParB networks.

scholarly journals Author response: ParB dynamics and the critical role of the CTD in DNA condensation unveiled by combined force-fluorescence measurements

2019 ◽  
Author(s):  
Julene Madariaga-Marcos ◽  
Cesar L Pastrana ◽  
Gemma LM Fisher ◽  
Mark Simon Dillingham ◽  
Fernando Moreno-Herrero
Keyword(s):  
Critical Role ◽  
Dna Condensation ◽  
Author Response ◽  
Fluorescence Measurements

scholarly journals Force determination in lateral magnetic tweezers combined with TIRF microscopy

Nanoscale ◽  
2018 ◽  
Vol 10 (9) ◽  
pp. 4579-4590 ◽  
Author(s):  
J. Madariaga-Marcos ◽  
S. Hormeño ◽  
C. L. Pastrana ◽  
G. L. M. Fisher ◽  
M. S. Dillingham ◽  
...  
Keyword(s):  
Dna Binding ◽  
Magnetic Tweezers ◽  
Constant Force ◽  
Dna Molecules ◽  
Tirf Microscopy

We have designed and calibrated a magnetic tweezers module to laterally stretch DNA molecules at a constant force, which can be incorporated into conventional magnetic tweezers. We demonstrate the combination of lateral magnetic tweezers with TIRF microscopy by characterizing DNA binding by ParB.

scholarly journals FANCJ (BACH1) helicase forms DNA damage inducible foci with replication protein A and interacts physically and functionally with the single-stranded DNA-binding protein

Blood ◽  
2007 ◽  
Vol 110 (7) ◽  
pp. 2390-2398 ◽  
Author(s):  
Rigu Gupta ◽  
Sudha Sharma ◽  
Joshua A. Sommers ◽  
Mark K. Kenny ◽  
Sharon B. Cantor ◽  
...  
Keyword(s):  
Dna Damage ◽  
Dna Binding ◽  
Binding Protein ◽  
Critical Role ◽  
Protein A ◽  
Replication Protein A ◽  
Dna Binding Protein ◽  
Replication Protein ◽  
Single Stranded Dna

The BRCA1 associated C-terminal helicase (BACH1, designated FANCJ) is implicated in the chromosomal instability genetic disorder Fanconi anemia (FA) and hereditary breast cancer. A critical role of FANCJ helicase may be to restart replication as a component of downstream events that occur during the repair of DNA cross-links or double-strand breaks. We investigated the potential interaction of FANCJ with replication protein A (RPA), a single-stranded DNA-binding protein implicated in both DNA replication and repair. FANCJ and RPA were shown to coimmunoprecipitate most likely through a direct interaction of FANCJ and the RPA70 subunit. Moreover, dependent on the presence of BRCA1, FANCJ colocalizes with RPA in nuclear foci after DNA damage. Our data are consistent with a model in which FANCJ associates with RPA in a DNA damage-inducible manner and through the protein interaction RPA stimulates FANCJ helicase to better unwind duplex DNA substrates. These findings identify RPA as the first regulatory partner of FANCJ. The FANCJ-RPA interaction is likely to be important for the role of the helicase to more efficiently unwind DNA repair intermediates to maintain genomic stability.

Mixed-ligand copper(ii) Schiff base complexes: the role of the co-ligand in DNA binding, DNA cleavage, protein binding and cytotoxicity

2016 ◽  
Vol 45 (22) ◽  
pp. 9073-9087 ◽  
Author(s):  
Wen-Jing Lian ◽  
Xin-Tian Wang ◽  
Cheng-Zhi Xie ◽  
He Tian ◽  
Xue-Qing Song ◽  
...  
Keyword(s):  
Schiff Base ◽  
Dna Binding ◽  
Protein Binding ◽  
Dna Cleavage ◽  
Mixed Ligand ◽  
Schiff Base Complexes ◽  
Biological Features

Four novel mixed-ligand copper(ii) Schiff base complexes were synthesized and characterized. The biological features of the complexes and how acetic auxiliary ligands manipulate these features were investigated.

A critical role in structure-specific DNA binding for the acetylatable lysine residues in HMGB1

2008 ◽  
Vol 411 (3) ◽  
pp. 553-561 ◽  
Author(s):  
René Assenberg ◽  
Michelle Webb ◽  
Edward Connolly ◽  
Katherine Stott ◽  
Matthew Watson ◽  
...  
Keyword(s):  
Dna Binding ◽  
Critical Role ◽  
High Mobility ◽  
Full Length ◽  
High Mobility Group Protein ◽  
Bent Dna ◽  
Double Mutation ◽  
Lysine Residues ◽  
Group Protein

The structure-specific DNA-binding protein HMGB1 (high-mobility group protein B1) which comprises two tandem HMG boxes (A and B) and an acidic C-terminal tail, is acetylated in vivo at Lys2 and Lys11 in the A box. Mutation to alanine of both residues in the isolated A domain, which has a strong preference for pre-bent DNA, abolishes binding to four-way junctions and 88 bp DNA minicircles. The same mutations in full-length HMGB1 also abolish its binding to four-way junctions, and binding to minicircles is substantially impaired. In contrast, when the acidic tail is absent (AB di-domain) there is little effect of the double mutation on four-way junction binding, although binding to minicircles is reduced ∼15-fold. Therefore it appears that in AB the B domain is able to substitute for the non-functional A domain, whereas in full-length HMGB1 the B domain is masked by the acidic tail. In no case does single substitution of Lys2 or Lys11 abolish DNA binding. The double mutation does not significantly perturb the structure of the A domain. We conclude that Lys2 and Lys11 are critical for binding of the isolated A domain and HMGB1 to distorted DNA substrates.

scholarly journals Myosin IIA–mediated forces regulate multicellular integrity during vascular sprouting

2019 ◽  
Vol 30 (16) ◽  
pp. 1974-1984 ◽  
Author(s):  
Christine Yoon ◽  
Colin Choi ◽  
Sarah Stapleton ◽  
Teodelinda Mirabella ◽  
Caroline Howes ◽  
...  
Keyword(s):  
Network Formation ◽  
Critical Role ◽  
Three Dimensional ◽  
Adherens Junction ◽  
Cell Junctions ◽  
Cell Contractility ◽  
Surrounding Matrix ◽  
Contractile Forces ◽  
Cell Cell

Angiogenic sprouting is a critical process involved in vascular network formation within tissues. During sprouting, tip cells and ensuing stalk cells migrate collectively into the extracellular matrix while preserving cell–cell junctions, forming patent structures that support blood flow. Although several signaling pathways have been identified as controlling sprouting, it remains unclear to what extent this process is mechanoregulated. To address this question, we investigated the role of cellular contractility in sprout morphogenesis, using a biomimetic model of angiogenesis. Three-dimensional maps of mechanical deformations generated by sprouts revealed that mainly leader cells, not stalk cells, exert contractile forces on the surrounding matrix. Surprisingly, inhibiting cellular contractility with blebbistatin did not affect the extent of cellular invasion but resulted in cell–cell dissociation primarily between tip and stalk cells. Closer examination of cell–cell junctions revealed that blebbistatin impaired adherens-junction organization, particularly between tip and stalk cells. Using CRISPR/Cas9-mediated gene editing, we further identified NMIIA as the major isoform responsible for regulating multicellularity and cell contractility during sprouting. Together, these studies reveal a critical role for NMIIA-mediated contractile forces in maintaining multicellularity during sprouting and highlight the central role of forces in regulating cell–cell adhesions during collective motility.

Mixed ligand copper(ii) dicarboxylate complexes: the role of co-ligand hydrophobicity in DNA binding, double-strand DNA cleavage, protein binding and cytotoxicity

2015 ◽  
Vol 44 (22) ◽  
pp. 10210-10227 ◽  
Author(s):  
Rangasamy Loganathan ◽  
Sethu Ramakrishnan ◽  
Mani Ganeshpandian ◽  
Nattamai S. P. Bhuvanesh ◽  
Mallayan Palaniandavar ◽  
...  
Keyword(s):  
Dna Binding ◽  
Protein Binding ◽  
Dna Cleavage ◽  
Mixed Ligand ◽  
Double Strand Dna

Mixed ligand 5,6-dmp and 3,4,7,8-tmp complexes display more prominent cytotoxicity and higher apoptotic inducing ability.

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