Long DNA constructs to study helicases and nucleic acid translocases using optical tweezers

Aicart-Ramos, Clara; Hormeño, Silvia; Wilkinson, Oliver; Dillingham, Mark S.; Moreno‐Herrero, Fernando

doi:10.1016/bs.mie.2022.03.010

Cited by 5 publications

(4 citation statements)

References 105 publications

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“…A DNA plasmid containing 16x OB sites (ATTTTAGCGGCTAAAAG) and 1x OA site (AATGTTTAGCTAAACCTT) was produced by modification of a pUC19 plasmid containing a single copy of each site separated by 1016 bp (pUC19_v1), following several cloning steps and methods described elsewhere 27,92 .…”

Section: Methodsmentioning

confidence: 99%

Molecular switching of a DNA-sliding clamp to a repressor mediates long-range gene silencing

McLean,

Balaguer-Perez,

Chandanani

et al. 2024

Preprint

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Long-range gene regulation is rare in bacteria and is confined to the classical DNA looping model. Here, we use a combination of biophysical approaches, including X-ray crystallography and single-molecule analysis, to show that long-range gene silencing on the plasmid RK2, a source of multidrug resistance across diverse Gram-negative bacteria, is achieved cooperatively by a DNA-sliding clamp, KorB, and a clamp-locking protein, KorA. We find that KorB is a CTPase clamp that can entrap and slide along DNA to reach distal target promoters. We resolved the tripartite crystal structure of a KorB-KorA-DNA co-complex, revealing that KorA latches KorB into a closed-clamp state. KorA thus stimulates repression by stalling KorB sliding at target promoters to occlude RNA polymerase holoenzymes. Altogether, our findings explain the mechanistic basis for KorB role-switching from a DNA-sliding clamp to a co-repressor, and provide a new paradigm for the long-range regulation of gene expression.

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Section: Methodsmentioning

confidence: 99%

Molecular switching of a DNA-sliding clamp to a repressor mediates long-range gene silencing

McLean,

Balaguer-Perez,

Chandanani

et al. 2024

Preprint

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“…The digested dsDNA duplex was ligated into the linearized large plasmid, resulting in a large plasmid with 3x parS sites (20622 bp). Plasmids were introduced into E. coli DH5α competent cells and potentially positive colonies were then selected by colony PCR (48). Plasmids were purified from the cultures using a QIAprep Spin Miniprep Kit (QIAGEN), analyzed by restriction enzyme digestion, and finally verified by Sanger sequencing.…”

Section: Methodsmentioning

confidence: 99%

“…C-trap experiments were performed on dsDNA molecules of 20622 bp containing 3x parS sites. The central part of the dsDNA construct was obtained by digestion of the large C-trap plasmid (see above) with NotI (NEB) produced following published protocols (48). Without further purification, the fragment was ligated to highly biotinylated handles of ∼1 kb ending in PspOMI.…”

Section: Methodsmentioning

confidence: 99%

Assembly of a segrosome by a CTP-independent ParB-like protein

Sukhoverkov,

Balaguer-Perez,

Aicart-Ramos

et al. 2024

Preprint

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The ATP- and CTP-dependent ParA-ParB-parS segrosome is a macromolecular complex that segregates chromosomes/plasmids in most bacterial species. CTP binding and hydrolysis enable ParB to slide on DNA and to bridge and condense DNA, thereby dictating the size and dynamics of the tripartite ParABS complex. Several other evolutionarily distinct systems can also segregate DNA, although the full diversity of bacterial DNA partition systems is not yet known. Here, we identify a CTP-independent ParABS system that maintains a conjugative plasmid SCP2 in the filamentous bacterium Streptomyces coelicolor. We demonstrate that an SCP2 ParB-like protein, ParT, loads onto DNA at an 18-bp parS site and diffuses away to the adjacent DNA despite lacking an apparent CTPase domain and detectable NTPase activity. We further show that parS DNA stimulates ParT transition from loading to a diffusing state to accumulate on DNA, and ParT activates the ATPase activity of its cognate partner protein ParA. We also identify numerous structural homologs of ParT, suggesting that CTP-independent diffusion on DNA might be widespread in bacteria despite being previously unappreciated. Overall, our findings uncover a CTP-independent DNA translocation as an alternative and unanticipated mechanism for the assembly of a bacterial DNA segregation complex and suggest that CTP binding and hydrolysis is not a fundamental feature of ParABS-like systems.

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“…DNA substrates -C-Trap experiments were performed on dsDNA molecules of 25427 bp. The central part of the DNA construct was obtained by digestion of a large homemade plasmid with BssHII (New England Biolabs) produced following published protocols (54). Without further purification, the fragment was ligated to highly biotinylated handles of ~1 kbp ending in MluI.…”

Section: Optical Tweezers and Confocal Microscopy Assaymentioning

confidence: 99%

DNA binding and bridging by human CtIP in the healthy and diseased states

Balaji,

De Bragança,

Balaguer-Pérez

et al. 2023

Preprint

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The human DNA repair factor CtIP helps to initiate the resection of double-stranded DNA breaks for repair by homologous recombination, in part through its ability to bind and bridge DNA molecules. However, CtIP is a natively disordered protein that bears no apparent similarity to other DNA-binding proteins and so the structural basis for these activities remains unclear. In this work, we have used bulk DNA binding, single molecule tracking, and DNA bridging assays to study wild-type and variant CtIP proteins to better define the DNA binding domains and the effects of mutations associated with inherited human disease. Our work identifies a monomeric DNA-binding domain in the C-terminal region of CtIP. CtIP binds non-specifically to DNA and can diffuse over thousands of nucleotides. CtIP-mediated bridging of distant DNA segments is observed in single-molecule magnetic tweezers experiments. However, we show that binding alone is insufficient for DNA bridging, which also requires tetramerization via the N-terminal domain. Variant CtIP proteins associated with Seckel and Jawad syndromes display impaired DNA binding and bridging activities. The significance of these findings in the context of facilitating DNA break repair is discussed.Significance StatementCtIP helps to repair broken chromosomes through its ability to bind and bridge DNA molecules. We studied the structural and biochemical basis for these activities and how they are affected by hereditary CtIP mutations associated with developmental disorders. We discovered a minimal domain in the C-terminal region of CtIP which supports DNA binding as a monomer. DNA binding is non-specific and facilitates 1D diffusion, but binding alone is insufficient for intermolecular tethering of DNA molecules which requires tetramerization of CtIP via N-terminal coiled-coil domains. All disease variants tested displayed impaired DNA bridging activity. These results have important implications for understanding the role of CtIP as a hub protein for DNA break repair and its dysfunction in human disease.

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Long DNA constructs to study helicases and nucleic acid translocases using optical tweezers

Cited by 5 publications

References 105 publications

Molecular switching of a DNA-sliding clamp to a repressor mediates long-range gene silencing

Molecular switching of a DNA-sliding clamp to a repressor mediates long-range gene silencing

Assembly of a segrosome by a CTP-independent ParB-like protein

DNA binding and bridging by human CtIP in the healthy and diseased states

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