Facilitates chromatin transcription (FACT) plays essential roles in chromatin remodeling during DNA transcription, replication, and repair. Our structural and biochemical studies of human FACT-histone interactions present precise views of nucleosome reorganization, conducted by the FACT-SPT16 (suppressor of Ty 16) Mid domain and its adjacent acidic AID segment. AID accesses the H2B N-terminal basic region exposed by partial unwrapping of the nucleosomal DNA, thereby triggering the invasion of FACT into the nucleosome. The crystal structure of the Mid domain complexed with an H3-H4 tetramer exhibits two separate contact sites; the Mid domain forms a novel intermolecular β structure with H4. At the other site, the Mid-H2A steric collision on the H2A-docking surface of the H3-H4 tetramer within the nucleosome induces H2A-H2B displacement. This integrated mechanism results in disrupting the H3 αN helix, which is essential for retaining the nucleosomal DNA ends, and hence facilitates DNA stripping from histone.

Gene expression in eukaryotes depends upon positioning, mobility and packaging of nucleosomes; thus, we need the detailed information of the human nucleosome core particle (NCP) structure, which could clarify chromatin properties. Here, we report the 2.5 Å crystal structure of a human NCP. The overall structure is similar to those of other NCPs reported previously. However, the DNA path of human NCP is remarkably different from that taken within other NCPs with an identical DNA sequence. A comparison of the structural parameters between human and Xenopus laevis DNA reveals that the DNA path of human NCP consecutively shifts by 1 bp in the regions of superhelix axis location −5.0 to −2.0 and 5.0 to 7.0. This alteration of the human DNA path is caused predominantly by tight DNA–DNA contacts within the crystal. It is also likely that the conformational change in the human H2B tail induces the local alteration of the DNA path. In human NCP, the region with the altered DNA path lacks Mn2+ ions and the B-factors of the DNA phosphate groups are substantially high. Therefore, in contrast to the histone octamer, the nucleosomal DNA is sufficiently flexible and mobile and can undergo drastic conformational changes, depending upon the environment.

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The atomic view of the active site coupling termed channelling is a major subject in molecular biology. We have determined two distinct crystal structures of the bacterial multienzyme complex that catalyzes the last three sequential reactions in the fatty acid b-oxidation cycle. The a 2 b 2 heterotetrameric structure shows the uneven ring architecture, where all the catalytic centers of 2-enoyl-CoA hydratase (ECH), L-3-hydroxyacyl-CoA dehydrogenase (HACD) and 3-ketoacyl-CoA thiolase (KACT) face a large inner solvent region. The substrate, anchored through the 3 0 -phosphate ADP moiety, allows the fatty acid tail to pivot from the ECH to HACD active sites, and finally to the KACT active site. Coupling with striking domain rearrangements, the incorporation of the tail into the KACT cavity and the relocation of 3 0 -phosphate ADP bring the reactive C2-C3 bond to the correct position for cleavage. The ahelical linker specific for the multienzyme contributes to the pivoting center formation and the substrate transfer through its deformation. This channelling mechanism could be applied to other b-oxidation multienzymes, as revealed from the homology model of the human mitochondrial trifunctional enzyme complex.

Intrinsically disordered (ID) regions of proteins are recognized to be involved in biological processes such as transcription, translation, and cellular signal transduction. Despite the important roles of ID regions, effective methods to observe these thin and flexible structures directly were not available. Herein, we use high-speed atomic force microscopy (AFM) to observe the heterodimeric FACT (facilitates chromatin transcription) protein, which is predicted to have large ID regions in each subunit. Successive AFM images of FACT on a mica surface, captured at rates of 5-17 frames per second, clearly reveal two distinct tail-like segments that protrude from the main body of FACT and fluctuate in position. Using deletion mutants of FACT, we identify these tail segments as the two major ID regions predicted from the amino acid sequences. Their mechanical properties estimated from the AFM images suggest that they have more relaxed structures than random coils. These observations demonstrate that this state-of-the-art microscopy method can be used to characterize unstructured protein segments that are difficult to visualize with other experimental techniques.

FACT is a heterodimer of SPT16 and SSRP1, which each contain several conserved regions in the primary structure. The interaction of FACT with nucleosomes induces chromatin remodeling through the combinatorial action of its distinct functional protein regions. However, there is little mechanistic insight into how these regions cooperatively contribute to FACT functions, particularly regarding the recognition of nucleosomal DNA. Here, we report the identification of novel phosphorylation sites of Drosophila melanogaster FACT (dFACT) expressed in Sf9 cells. These sites are densely concentrated in the acidic intrinsically disordered (ID) region of the SSRP1 subunit and control nucleosomal DNA binding by dFACT. This region and the adjacent segment of the HMG domain form weak electrostatic intramolecular interactions, which is reinforced by the phosphorylation, thereby blocking DNA binding competitively. Importantly, this control mechanism appears to support rapid chromatin transactions during early embryogenesis through the dephosphorylation of some sites in the maternally transmitted dSSRP1. FACT (facilitates chromatin transcription),2 an evolutionarily conserved protein in eukaryotes, is a heterodimer consisting of structure-specific recognition protein-1 (SSRP1) and SPT16 with a larger molecular mass than SSRP1 (1, 2). FACT is classified as a chromatin-remodeling factor essential for various processes within nuclei, such as transcription, DNA replication, and DNA repair (1, 3-11). In the transcriptional process, FACT displaces histone H2A/H2B dimers from nucleosomes, thereby facilitating RNA polymerase II transcription (12). The FACT subunits also display a range of physical and genetic interactions with other factors (2,3,6,11,13), suggesting that FACT directs several different functions by interacting with multiple complexes.At the molecular level, FACT initially binds nucleosomes and/or nucleosomal DNA, and then destabilizes the interactions between the H2A/H2B dimers and the H3/H4 tetramer within nucleosomes (2). Therefore, most studies have so far focused on interactions between FACT and histones. For example, it has been previously reported that the C-terminal region of human SPT16 (hSPT16) directly binds to H2A/H2B dimers (12). A recent study has revealed that the N-terminal amino peptidase-like domain of Schizosaccharomyces pombe SPT16 associates with the H3/H4 histones, suggesting that this SPT16 may contribute to binding, eviction, and/or deposition of all histones (14). However, it remains unclear how the FACT protein interacts with the nucleosomal DNA at the initial step of chromatin remodeling, although several studies have reported that the high-mobility group (HMG) box domain of SSRP1 binds to DNA nonspecifically or by recognizing specific structures of DNA (15-17).The heterodimeric FACT complex consists of several distinct structural domains and intrinsically disordered (ID) regions (Fig. 1A). The functional aspects of these folded domains or unstructured regions have not been clarified yet. The sm...

We have analyzed the cleavage speci¢cities of various prokaryotic Type 2 ribonucleases H (RNases H) on chimeric DNA^RNA^DNA/DNA substrates containing one to four ribonucleotides. RNases HII from Bacillus subtilis and Thermococcus kodakaraensis cleaved all of these substrates to produce a DNA segment with a 5P P-monoribonucleotide. Consequently, these enzymes cleaved even the chimeric substrate containing a single ribonucleotide at the DNA^RNA junction (5P P-side of the single ribonucleotide). In contrast, Escherichia coli RNase HI and B. subtilis RNase HIII did not cleave the chimeric substrate containing a single ribonucleotide. These results suggest that bacterial and archaeal RNases HII are involved in excision of a single ribonucleotide misincorporated into DNA.

Facilitates chromatin transcription (FACT) is a histone chaperone, which accomplishes both nucleosome assembly and disassembly. Our combined cryo-electron microscopy (EM) and native mass spectrometry (MS) studies revealed novel key steps of nucleosome reorganization conducted by a Mid domain and its adjacent acidic AID segment of human FACT. We determined three cryo-EM structures of respective octasomes complexed with the Mid-AID and AID regions, and a hexasome alone. We discovered extensive contacts between a FACT region and histones H2A, H2B, and H3, suggesting that FACT is competent to direct functional replacement of a nucleosomal DNA end by its phosphorylated AID segment (pAID). Mutational assays revealed that the aromatic and phosphorylated residues within pAID are essential for octasome binding. The EM structure of the hexasome, generated by the addition of Mid-pAID or pAID, indicated that the dissociation of H2A-H2B dimer causes significant alteration from the canonical path of the nucleosomal DNA.