Arrays of regularly spaced nucleosomes directly correlate with closed chromatin structures at silenced loci. The ATP-dependent chromatin-assembly factor (ACF) generates such arrays in vitro and is required for transcriptional silencing in vivo. A key unresolved question is how ACF 'measures' equal spacing between nucleosomes. We show that ACF senses flanking DNA length and transduces length information in an ATP-dependent manner to regulate the rate of nucleosome movement. Using fluorescence resonance energy transfer to follow nucleosome movement, we find that ACF can rapidly sample DNA on either side of a nucleosome and moves the longer flanking DNA across the nucleosome faster than the shorter flanking DNA. This generates a dynamic equilibrium in which nucleosomes having equal DNA on either side accumulate. Our results indicate that ACF generates the characteristic 50- to 60-base-pair internucleosomal spacing in silent chromatin by kinetically discriminating against shorter linker DNAs.

The ATP-utilizing chromatin assembly and remodelling factor (ACF) functions to generate regularly spaced nucleosomes, which are required for heritable gene silencing. The mechanism by which ACF mobilizes nucleosomes remains poorly understood. Here we report a single-molecule FRET study that monitors the remodelling of individual nucleosomes by ACF in real time, revealing previously unknown remodelling intermediates and dynamics. In the presence of ACF and ATP, the nucleosomes exhibit gradual translocation along DNA interrupted by well-defined kinetic pauses that occurred after approximately 7 or 3 – 4 base pairs of translocation. The binding of ACF, translocation of DNA, and exiting of translocation pauses are all ATP-dependent, revealing three distinct functional roles of ATP during remodelling. At equilibrium, a continuously bound ACF complex can move the nucleosome back-and-forth many times before dissociation, indicating that ACF is a highly processive and bidirectional nucleosome translocase.

Evenly spaced nucleosomes directly correlate with condensed chromatin and gene silencing. The ATP-dependent chromatin assembly factor (ACF) forms such structures in vitro and is required for silencing in vivo. ACF generates and maintains nucleosome spacing by constantly moving a nucleosome towards the longer flanking DNA faster than the shorter flanking DNA. But how the enzyme rapidly moves back and forth between both sides of a nucleosome to accomplish bidirectional movement is unknown. We show that nucleosome movement depends cooperatively on two ACF molecules, suggesting that ACF functions as a dimer of ATPases. Further, the nucleotide state determines whether the dimer closely engages one vs. both sides of the nucleosome. Three-dimensional reconstruction by single particle electron microscopy of the ATPase-nucleosome complex in an activated ATP state reveals a dimer architecture in which the two ATPases face each other. Our results suggest a model in which the two ATPases work in a coordinated manner, taking turns to engage either side of a nucleosome, thereby allowing processive bidirectional movement. This novel dimeric motor mechanism differs from that of dimeric motors such as kinesin and dimeric helicases that processively translocate unidirectionally and reflects the unique challenges faced by motors that move nucleosomes.Chromatin-remodeling motors play essential roles in organizing the chromatin state for regulating eukaryotic genomes, yet how they carry out their myriad activities is poorly understood. Their substrate, the nucleosome, contains 147 bp of DNA wrapped in ~1.5 turns Users may view, print, copy, download and text and data-mine the content in such documents, for the purposes of academic research, subject always to the full Conditions of use: http://www.nature.com/authors/editorial_policies/license.html#terms around an octamer of histone proteins. Even the smallest movement of the histone octamer relative to the DNA presumably requires a coordinated process of breaking and reforming the many histone-DNA contacts. The ACF chromatin-remodeling complex exemplifies the task, as it is able to move nucleosomes to create evenly spaced nucleosomal arrays that contain equal DNA on either side of each nucleosome1-10. These evenly spaced arrays are important for packaging the underlying DNA into silent chromatin structures in vivo1-10. HHS Public AccessACF is part of the ISWI family of remodeling complexes. The ATPase subunits of ISWI complexes can move nucleosomes by themselves while the accessory subunits modulate this basic activity11-15. The human ACF complex consists of one ATPase subunit, SNF2h and one accessory subunit, Acf16,7. SNF2h is part of the SF2 family of DExx box proteins that includes helicases and nucleic acid translocases16. The ATPase domain of SNF2h has two RecA-like domains, which are thought to form a cleft within which ATP binds. SNF2h also has an alpha-helical extension comprised of three additional domains, HAND, SANT and SLIDE which are thought to play a role in bi...

While significant progress has been achieved in the structural analysis of exporters from the superfamily of ATP Binding Cassette (ABC) transporters, much less is known about how they selectively recognize substrates and how substrate binding is coupled to ATP hydrolysis. We have addressed these questions through the crystallographic analysis at 2.4 Å resolution of the Atm1/ABCB7/HMT1/ABCB6 ortholog from Novosphingobium aromaticivorans DSM 12444. Consistent with a physiological role in cellular detoxification processes, functional studies demonstrate that glutathione derivatives can serve as substrates for NaAtm1 and overexpression in E. coli confers protection against silver and mercury toxicity. The glutathione binding site highlights the articulated design of ABC exporters, with ligands and nucleotides spanning structurally conserved elements to create adaptable interfaces accommodating conformational rearrangements during the transport cycle.

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Acinetobacter baumannii is an aerobic and gram-negative pathogenic bacterium that is resistant to most antibiotics. Recently, A. baumannii 1656-2 exhibited the ability to form biofilms under clinical conditions. In this study, global metabolite profiling of both planktonic and biofilm forms of A. baumannii 1656-2 was performed using high-resolution nuclear magnetic resonance (NMR) spectroscopy and multivariate statistical analysis to investigate the metabolic patterns leading to biofilm formation. Principal components analysis (PCA) and orthogonal partial least-squares discriminant analysis (OPLS-DA) score plots showed a distinct separation between planktonic and biofilm cells. Metabolites including acetates, pyruvate, succinate, UDP-glucose, AMP, glutamate, and lysine were increasingly involved in the energy metabolism of biofilm formation. In particular, the ratio of N-acetyl-D-glucosamine (GlcNAc) to D-glucosamine (GlcNH2) was significantly higher during biofilm formation than under the planktonic condition. This study demonstrates that NMR-based global metabolite profiling of bacterial cells can provide valuable insight into the metabolic changes in multidrug resistant and biofilm-forming bacteria such as A. baumannii 1656-2.

Ginsenoside-Rd may be of some benefit in acute ischaemic stroke.