Single-molecule kinetics reveal microscopic mechanism by which High-Mobility Group B proteins alter DNA flexibility

McCauley, Micah J.; Rueter, Emily M.; Rouzina, Ioulia; Maher, L. James; Williams, Mark C.

doi:10.1093/nar/gks1031

Cited by 51 publications

(99 citation statements)

References 72 publications

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“…Some studies have shown that DNA binding by HMGB1 is dynamic in vitro [30,31] and also in cells [32], while other studies measured slower rates of dissociation [33]. A recent single molecule kinetic study using optical tweezers proposed that microscopic and macroscopic dissociation constants control interactions between HMGB proteins and DNA [34]. The microscopic dissociation events are rapid and involve breaking short range protein-DNA contacts, while the macroscopic events can be measured as slow depending on the experimental conditions, and involve complete dissociation and escape of the protein into solution.…”

Section: Discussionmentioning

confidence: 99%

The HMGB1 C-Terminal Tail Regulates DNA Bending

Blair

Horn

Pazhani

et al. 2016

Journal of Molecular Biology

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HMGB1 (high mobility group box protein 1) is an architectural protein that facilitates formation of protein-DNA assemblies involved in transcription, recombination, DNA repair, and chromatin remodeling. Important to its function is the ability of HMGB1 to bend DNA non-sequence specifically. HMGB1 contains two HMG boxes that bind and bend DNA (the A box and the B box) and a C-terminal acidic tail. We investigated how these domains contribute to DNA bending by HMGB1 using single molecule FRET, which enabled us to resolve heterogeneous populations of bent and unbent DNA. We found that full length HMGB1 bent DNA more than the individual A and B boxes. Removing the C-terminal tail resulted in a protein that bent DNA to a greater extent than the full length protein. These data suggest that the A and B boxes simultaneously bind DNA in the absence of the C-terminal tail, but the tail modulates DNA binding and bending by one of the HMG boxes in the full length protein. Indeed, a construct composed of the B box and the C-terminal tail only bent DNA at higher protein concentrations. Moreover, in the context of the full length protein, mutating the A box such that it could not bend DNA resulted in a protein that bent DNA similarly to a single HMG box and only at higher protein concentrations. We propose a model in which the HMGB1 C-terminal tail serves as an intramolecular damper that modulates the interaction of the B box with DNA.

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Section: Discussionmentioning

confidence: 99%

The HMGB1 C-Terminal Tail Regulates DNA Bending

Blair

Horn

Pazhani

et al. 2016

Journal of Molecular Biology

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“…It is of interest to consider the situation where one has a long dsDNA subject to insertion of kinks when proteins bind to it; this situation has been studied in a variety of single-DNA experiments [50, 51, 52, 53, 54] and is a simplified version of the situation occurring in vivo . A simple polymer model that is useful for considering this situation is a discretized semiflexible chain model, where each segment is a potential binding site for a protein, which when present induces a bend [55]:…”

Section: Dna-protein Interactionsmentioning

confidence: 99%

Biophysics of protein–DNA interactions and chromosome organization

Marko

2015

Physica A: Statistical Mechanics and its Applications

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The function of DNA in cells depends on its interactions with protein molecules, which recognize and act on base sequence patterns along the double helix. These notes aim to introduce basic polymer physics of DNA molecules, biophysics of protein-DNA interactions and their study in single-DNA experiments, and some aspects of large-scale chromosome structure. Mechanisms for control of chromosome topology will also be discussed.

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“…Alternatively, rapidly mixing excess DNA that contain the TF target site can be used to quickly deplete the concentration of unbound TF. However, addition of competitor DNA can bind with partially bound TF states and result in observed dissociation rates that are much higher than the inherent rate of TF dissociation [30–33]. …”

Section: Experimental Designmentioning

confidence: 99%

Single molecule fluorescence methodologies for investigating transcription factor binding kinetics to nucleosomes and DNA

Luo

North

Poirier

2014

Methods

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Site specific DNA binding complexes must bind their DNA target sites and then reside there for a sufficient amount of time for proper regulation of DNA processing including transcription, replication and DNA repair. In eukaryotes, the occupancy of DNA binding complexes at their target sites is regulated by chromatin structure and dynamics. Methodologies that probe both the binding and dissociation kinetics of DNA binding proteins with naked and nucleosomal DNA are essential for understanding the mechanisms by which these complexes function. Here, we describe single-molecule fluorescence methodologies for quantifying the binding and dissociation kinetics of transcription factors at a target site within DNA, nucleosomes and nucleosome arrays. This approach allowed for the unexpected observation that nucleosomes impact not only binding but also dissociation kinetics of transcription factors and is well-suited for the investigation of numerous DNA processing complexes that directly interact with DNA organized into chromatin.

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Single-molecule kinetics reveal microscopic mechanism by which High-Mobility Group B proteins alter DNA flexibility

Cited by 51 publications

References 72 publications

The HMGB1 C-Terminal Tail Regulates DNA Bending

The HMGB1 C-Terminal Tail Regulates DNA Bending

Biophysics of protein–DNA interactions and chromosome organization

Single molecule fluorescence methodologies for investigating transcription factor binding kinetics to nucleosomes and DNA

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