Vital insights into prokaryotic genome compaction by nucleoid-associated protein (NAP) and illustration of DNA flexure angles at single-molecule resolution

Purkait, Debayan; Bandyopadhyay, Debolina; Mishra, Padmaja P.

doi:10.1016/j.ijbiomac.2020.12.194

Cited by 6 publications

(3 citation statements)

References 77 publications

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“…In the context of DNA duplex bending, WtIHF is well-known for facilitating essential cellular processes, including site-specific recombination, replication, transcription, and DNA compaction. Recent SmFRET studies (Purkait et al , 2021), atomic force microscopy investigations, and molecular dynamics simulations have shed light on the multimodal bending of DNA duplexes induced by IHF (Yoshua et al , 2021). IHF-induced DNA bending has been a well-established concept stemming from structural studies.…”

Section: Resultsmentioning

confidence: 99%

Unveiling the Holliday junction conformational dynamics of upon binding with DNA architectural proteins IHF; A Single molecule FRET approach

Islam,

Mishra

2023

Preprint

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Integration host factor (IHF) is a vital nucleoid-associated protein that binds strongly to a 13-base-pair consensus sequence, inducing a significant bend of approximately 160° upon binding. The bending of DNA by Wild type IHF, aids the integration of foreign DNA into the host genome. Conversely, the Single Chain IHF (ScIHF) is designed for specific genetic engineering and biotechnology applications. We quantified IHF binding to Holliday junctions (HJs) containing specific binding sequences (H' & H1). Both IHF variants exhibited high-affinity binding to HJs in the presence of the consensus sequence, suggesting a structure-based recognition mechanism. HJs are dynamic, with open or stacked conformations facilitating processes like branch migration and strand exchange. The results highlighted protein's impact on junction conformation, which was further validated using smFRET. Our results show that both WtIHF and ScIHF shifted the population toward the open conformation of the junction and stabilized it in that state. In summary, our findings reveal that IHF binds HJs with high affinity and exerts a stabilizing influence on maintaining the open conformation of these DNA structures.

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Section: Resultsmentioning

confidence: 99%

Unveiling the Holliday junction conformational dynamics of upon binding with DNA architectural proteins IHF; A Single molecule FRET approach

Islam,

Mishra

2023

Preprint

Self Cite

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“…67 In the context of DNA duplex bending, WtIHF is well-known for facilitating essential cellular processes, including site-specific recombination, replication, transcription, and DNA compaction. Recent SmFRET studies, 68 atomic force microscopy investigations, and molecular dynamics simulations have shed light on the multimodal bending of DNA duplexes induced by IHF. 67 IHF-induced DNA bending has been a well-established concept, derived from structural studies.…”

Section: The Journal Ofmentioning

confidence: 99%

Molecular Insight into the Structural Dynamics of Holliday Junctions Modulated by Integration Host Factor

Islam,

Mishra

2024

J. Phys. Chem. B

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The integration host factor (IHF) in Escherichia coli is a nucleoid-associated protein with multifaceted roles that encompass DNA packaging, viral DNA integration, and recombination. IHF binds to double-stranded DNA featuring a 13-base pair (bp) consensus sequence with high affinity, causing a substantial bend of approximately 160°upon binding. Although wild-type IHF (WtIHF) is principally involved in DNA bending to facilitate foreign DNA integration into the host genome, its engineered counterpart, single-chain IHF (ScIHF), was specifically designed for genetic engineering and biotechnological applications. Our study delves into the interactions of both IHF variants with Holliday junctions (HJs), pivotal intermediates in DNA repair, and homologous recombination. HJs are dynamic structures capable of adopting open or stacked conformations, with the open conformation facilitating processes such as branch migration and strand exchange. Using microscale thermophoresis, we quantitatively assessed the binding of IHF to four-way DNA junctions that harbor specific binding sequences H' and H1. Our findings demonstrate that both IHF variants exhibit a strong affinity for HJs, signifying a structure-based recognition mechanism. Circular dichroism (CD) experiments unveiled the impact of the protein on the junction's conformation. Furthermore, single-molecule Forster resonance energy transfer (smFRET) confirmed the influence of IHF on the junction's dynamicity. Intriguingly, our results revealed that WtIHF and ScIHF binding shifts the population toward the open conformation of the junction and stabilizes it in that state. In summary, our findings underscore the robust affinity of the IHF for HJs and its capacity to stabilize the open conformation of these junctions.

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“…They include those containing DNA such as the protein complexes involved in DNA replication and segregation (Sunako et al, 2001)(Boeneman et al, 2009)(Duderstadt et al, 2010)(Sanchez-Romero et al, 2011)(Helgesen et al, 2015), complexes involving topoisomerases (McKie et al, 2021) along with RNA polymerase supramolecular complexes associated with topoisomerases and metabolic enzymes (Muskhelishvili et al, 2021). In the case of the nucleoid-associated proteins in E. coli, (1) H-NS has been reported not only to form hyperstructures (Wang et al, 2011)(Kuwada et al, 2015)(Gao et al, 2017) (for artefacts see(Margolin, 2012)) but also to help create "speci c mesoscale domains in chromosomal regions in response to environmental changes"(Cristofalo et al, 2020), (2) HU has been proposed to undergo a transition from a DNA bundle to a lament (depending both on the HU to DNA ratio and on environmental factors) that would alter the structure of the nucleoid(Hammel et al, 2016)(Remesh et al, 2020) (3) IHF has been proposed to bind to DNA to provide a 'nucleation point' for higher-order genome organisation(Purkait et al, 2021) that would involve HU and H-NS along with structural maintenance of chromosome (SMC or condensin) proteins. Both IHF and HU participate in pre-replication complexes at the E. coli oriC(Ryan et al, 2002).…”

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confidence: 99%

Hypothesis: bacteria live on the edge of phase transitions with a cell cycle regulated by a water-clock

2024

Preprint

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A fundamental problem in biology is how cells obtain the reproducible, coherent phenotypes needed for natural selection to act or, put differently, how cells manage to limit their exploration of the vastness of phenotype space. A subset of this problem is how they regulate their cell cycle. Bacteria, like eukaryotic cells, are highly structured and contain scores of hyperstructures or assemblies of molecules and macromolecules. The existence and functioning of certain of these hyperstructures depend on phase transitions. Here, I propose a conceptual framework to facilitate the development of water-clock hypotheses in which cells use water to generate phenotypes by living ‘on the edge of phase transitions’. I give an example of such a hypothesis in the case of the bacterial cell cycle and show how it offers a relatively novel ‘view from here’ that brings together a range of different findings about hyperstructures, phase transitions and water and that can be integrated with other hypotheses about differentiation, metabolism and the origins of life.

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Vital insights into prokaryotic genome compaction by nucleoid-associated protein (NAP) and illustration of DNA flexure angles at single-molecule resolution

Cited by 6 publications

References 77 publications

Unveiling the Holliday junction conformational dynamics of upon binding with DNA architectural proteins IHF; A Single molecule FRET approach

Unveiling the Holliday junction conformational dynamics of upon binding with DNA architectural proteins IHF; A Single molecule FRET approach

Molecular Insight into the Structural Dynamics of Holliday Junctions Modulated by Integration Host Factor

Hypothesis: bacteria live on the edge of phase transitions with a cell cycle regulated by a water-clock

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