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      Facilitated dissociation of transcription factors from single DNA binding sites

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          Abstract

          <p id="d5485972e269">Transcription factors (TFs) control biological processes by binding and unbinding to DNA. Therefore, it is crucial to understand the mechanisms that affect TF binding kinetics. Recent studies challenge the standard picture of TF binding kinetics by showing cases of proteins in solution accelerating TF dissociation rates through a facilitated dissociation (FD) process. Our study shows that FD can occur at the level of single binding sites without the action of large protein clusters or long DNA segments. Our results quantitatively support a model of FD in which competitor proteins invade partially dissociated states of DNA-bound TFs. FD is expected to be a general mechanism for modulating gene expression by altering the occupancy of TFs on the genome. </p><p class="first" id="d5485972e272">The binding of transcription factors (TFs) to DNA controls most aspects of cellular function, making the understanding of their binding kinetics imperative. The standard description of bimolecular interactions posits that TF off rates are independent of TF concentration in solution. However, recent observations have revealed that proteins in solution can accelerate the dissociation of DNA-bound proteins. To study the molecular basis of facilitated dissociation (FD), we have used single-molecule imaging to measure dissociation kinetics of Fis, a key <i>Escherichia coli</i> TF and major bacterial nucleoid protein, from single dsDNA binding sites. We observe a strong FD effect characterized by an exchange rate <span class="inline-formula"> <math id="i1" overflow="scroll"> <mrow> <mo>∼</mo> <mn>1</mn> <mo>×</mo> <msup> <mrow> <mn>10</mn> </mrow> <mn>4</mn> </msup> <mo> </mo> <msup> <mtext>M</mtext> <mrow> <mo>−</mo> <mn>1</mn> </mrow> </msup> <msup> <mtext>s</mtext> <mrow> <mo>−</mo> <mn>1</mn> </mrow> </msup> </mrow> </math> </span>, establishing that FD of Fis occurs at the single-binding site level, and we find that the off rate saturates at large Fis concentrations in solution. Although spontaneous (i.e., competitor-free) dissociation shows a strong salt dependence, we find that FD depends only weakly on salt. These results are quantitatively explained by a model in which partially dissociated bound proteins are susceptible to invasion by competitor proteins in solution. We also report FD of NHP6A, a yeast TF with structure that differs significantly from Fis. We further perform molecular dynamics simulations, which indicate that FD can occur for molecules that interact far more weakly than those that we have studied. Taken together, our results indicate that FD is a general mechanism assisting in the local removal of TFs from their binding sites and does not necessarily require cooperativity, clustering, or binding site overlap. </p>

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          Dynamic regulation of transcriptional states by chromatin and transcription factors.

          The interaction of regulatory proteins with the complex nucleoprotein structures that are found in mammalian cells involves chromatin reorganization at multiple levels. Mechanisms that support these transitions are complex on many timescales, which range from milliseconds to minutes or hours. In this Review, we discuss emerging concepts regarding the function of regulatory elements in living cells. We also explore the involvement of these dynamic and stochastic processes in the evolution of fluctuating transcriptional activity states that are now commonly reported in eukaryotic systems.
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            Transcriptional regulation of human DNA repair genes following genotoxic stress: trigger mechanisms, inducible responses and genotoxic adaptation

            DNA repair is the first barrier in the defense against genotoxic stress. In recent years, mechanisms that recognize DNA damage and activate DNA repair functions through transcriptional upregulation and post-translational modification were the focus of intensive research. Most DNA repair pathways are complex, involving many proteins working in discrete consecutive steps. Therefore, their balanced expression is important for avoiding erroneous repair that might result from excessive base removal and DNA cleavage. Amelioration of DNA repair requires both a fine-tuned system of lesion recognition and transcription factors that regulate repair genes in a balanced way. Transcriptional upregulation of DNA repair genes by genotoxic stress is counteracted by DNA damage that blocks transcription. Therefore, induction of DNA repair resulting in an adaptive response is only visible through a narrow window of dose. Here, we review transcriptional regulation of DNA repair genes in normal and cancer cells and describe mechanisms of promoter activation following genotoxic exposures through environmental carcinogens and anticancer drugs. The data available to date indicate that 25 DNA repair genes are subject to regulation following genotoxic stress in rodent and human cells, but for only a few of them, the data are solid as to the mechanism, homeostatic regulation and involvement in an adaptive response to genotoxic stress.
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              Concentration-Dependent Exchange of Replication Protein A on Single-Stranded DNA Revealed by Single-Molecule Imaging

              Replication protein A (RPA) is a ubiquitous eukaryotic single-stranded DNA (ssDNA) binding protein necessary for all aspects of DNA metabolism involving an ssDNA intermediate, including DNA replication, repair, recombination, DNA damage response and checkpoint activation, and telomere maintenance [1], [2], [3]. The role of RPA in most of these reactions is to protect the ssDNA until it can be delivered to downstream enzymes. Therefore a crucial feature of RPA is that it must bind very tightly to ssDNA, but must also be easily displaced from ssDNA to allow other proteins to gain access to the substrate. Here we use total internal reflection fluorescence microscopy and nanofabricated DNA curtains to visualize the behavior of Saccharomyces cerevisiae RPA on individual strands of ssDNA in real-time. Our results show that RPA remains bound to ssDNA for long periods of time when free protein is absent from solution. In contrast, RPA rapidly dissociates from ssDNA when free RPA is present in solution allowing rapid exchange between the free and bound states. In addition, the S. cerevisiae DNA recombinase Rad51 and E. coli single-stranded binding protein (SSB) also promote removal of RPA from ssDNA. These results reveal an unanticipated exchange between bound and free RPA suggesting a binding mechanism that can confer exceptionally slow off rates, yet also enables rapid displacement through a direct exchange mechanism that is reliant upon the presence of free ssDNA-binding proteins in solution. Our results indicate that RPA undergoes constant microscopic dissociation under all conditions, but this is only manifested as macroscopic dissociation (i.e. exchange) when free proteins are present in solution, and this effect is due to mass action. We propose that the dissociation of RPA from ssDNA involves a partially dissociated intermediate, which exposes a small section of ssDNA allowing other proteins to access to the DNA.
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                Author and article information

                Journal
                Proceedings of the National Academy of Sciences
                Proc Natl Acad Sci USA
                Proceedings of the National Academy of Sciences
                0027-8424
                1091-6490
                April 18 2017
                April 18 2017
                : 114
                : 16
                : E3251-E3257
                Article
                10.1073/pnas.1701884114
                5402408
                28364020
                f4e24343-d2b4-4ff2-a2ba-1ad2e7962b60
                © 2017

                http://www.pnas.org/site/misc/userlicense.xhtml

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