In preparation for bidirectional replication, the origin recognition complex (ORC) loads two MCM helicases forming a head-to-head double hexamer (DH) around DNA 1,2. How DH formation occurs is debated. Single-molecule experiments suggest a sequential mechanism whereby ORCdependent loading of the first hexamer drives second hexamer recruitment 3. In contrast, biochemical data show that two rings are loaded independently via the same ORC-mediated mechanism, at two inverted DNA sites 4,5. We visualized MCM loading using time-resolved EM, to identify DH formation intermediates. We confirm that both hexamers are recruited via the same interaction between the MCM and ORC C-terminal domains, and identify the mechanism for coupled MCM loading. A first loaded hexamer locked around DNA is recognized by ORC, which unexpectedly engages the N-terminal homo-dimerization interface of MCM. In this configuration, ORC is poised to direct second hexamer recruitment in an inverted orientation, suitable for DH formation. Our data reconcile two apparently contrasting models. Users may view, print, copy, and download text and data-mine the content in such documents, for the purposes of academic research, subject always to the full Conditions of use:

The polycomb group (PcG) proteins are a large and diverse family that epigenetically repress the transcription of key developmental genes. They form three broad groups of polycomb repressive complexes (PRCs) known as PRC1, PRC2 and Polycomb Repressive DeUBiquitinase, each of which modifies and/or remodels chromatin by distinct mechanisms that are tuned by having variable compositions of core and accessory subunits. Until recently, relatively little was known about how the various PcG proteins assemble to form the PRCs; however, studies by several groups have now allowed us to start piecing together the PcG puzzle. Here, we discuss some highlights of recent PcG structures and the insights they have given us into how these complexes regulate transcription through chromatin.

contributed equally to this work. Conflict of interest:The authors have declared that no conflict of interest exists. Nonstandard abbreviations used: tissue factor (TF); murine hepatitis virus type 3 (MHV-3); postcoitus (pc); alanine aminotransferase (ALT); hepatitis B surface antigen (HBsAg); hepatitis B e-antigen (HBeAg); hepatitis B viral capsid (HBcAg); severe acute respiratory syndrome (SARS).

2-Bromo-5,6-dichloro-1-β-d-ribofuranosyl benzimidazole (BDCRB) is a member of a new class of benzimidazole ribonucleosides which inhibit human cytomegalovirus (HCMV) late in the replication cycle without inhibiting viral DNA synthesis. We show here that polygenomic concatemeric HCMV DNA does not mature to unit genome length in the presence of BDCRB. To discover the locus of action, virus resistant to BDCRB was selected by serial passage in the presence of the compound. Genetic mapping experiments with BDCRB-resistant virus demonstrated that the resistance phenotype mapped to one amino acid (Asp344Glu; low resistance) or two amino acids (Asp344Glu and Ala355Thr; high resistance) within the product of exon 2 of the HCMV UL89 open reading frame. The HCMV UL89 open reading frame and its homologs are among the most conserved open reading frames in the herpesviruses, and their products have sequence similarities to a known ATP-dependent endonuclease from the double-stranded DNA bacteriophage T4. These findings strongly suggest that BDCRB prevents viral DNA maturation by interacting with a UL89 gene product and that the UL89 open reading frame may encode an endonucleolytic subunit of the putative HCMV terminase. Further, since mammalian cell DNA replication does not involve a DNA maturation step, compounds which inhibit viral DNA maturation should be selective and safe.

Bromodomains are critical components of many chromatin modifying/remodelling proteins and are emerging therapeutic targets, yet how they interact with nucleosomes, rather than acetylated peptides, remains unclear. Using BRDT as a model, we characterized how the BET family of bromodomains interacts with site-specifically acetylated nucleosomes. Here we report that BRDT interacts with nucleosomes through its first (BD1), but not second (BD2) bromodomain, and that acetylated histone recognition by BD1 is complemented by a bromodomain–DNA interaction. Simultaneous DNA and histone recognition enhances BRDT's nucleosome binding affinity and specificity, and its ability to localize to acetylated chromatin in cells. Conservation of DNA binding in bromodomains of BRD2, BRD3 and BRD4, indicates that bivalent nucleosome recognition is a key feature of these bromodomains and possibly others. Our results elucidate the molecular mechanism of BRDT association with nucleosomes and identify structural features of the BET bromodomains that may be targeted for therapeutic inhibition.

The objective of this study was to evaluate the neuropsychiatric effects of the alpha-2a adrenergic agonist guanfacine in children with Tourette syndrome (TS). Twenty-four children with TS participated in a 4-week, double-blind, placebo-controlled study of guanfacine. Tic severity, neuropsychologic functioning, and parent ratings of behavior were evaluated pre- and post-treatment. The sample had mild tic severity and subtle neuropsychologic dysfunction pretreatment. Post-treatment, patients receiving guanfacine were rated by parents as significantly improved (compared to placebo) on one measure of executive function (parent-rated metacognition). Improvement on tic severity, performance-based neuropsychologic measures, and all other parent ratings were not significantly better than placebo. At a moderate dose and short-term treatment duration, guanfacine did not provide significant neuropsychiatric benefits in this group of children with mild TS.

The Zn-coordinated PHD fingers of Pygopus (Pygo) proteins are critical for β-catenin-dependent transcriptional switches in normal and malignant tissues. They bind to methylated histone H3 tails, assisted by their BCL9 co-factors whose homology domain 1 (HD1) binds to the rear PHD surface. Although histone-binding residues are identical between the two human Pygo paralogs, we show here that Pygo2 complexes exhibit slightly higher binding affinities for methylated histone H3 tail peptides than Pygo1 complexes. We solved the crystal structure of the Pygo2 PHD–BCL9-2 HD1 complex, which revealed paralog-specific interactions in its PHD–HD1 interface that could contribute indirectly to its elevated affinity for the methylated histone H3 tail. Interestingly, using NMR spectroscopy, we discovered that HD1 binding to PHD triggers an allosteric communication with a conserved isoleucine residue that lines the binding channel for histone H3 threonine 3 (T3), the link between the two adjacent binding pockets accommodating histone H3 alanine 1 and methylated lysine 4, respectively. This modulates the surface of the T3 channel, providing a plausible explanation as to how BCL9 co-factors binding to Pygo PHD fingers impact indirectly on their histone binding affinity. Intriguingly, this allosteric modulation of the T3 channel is propagated through the PHD structural core by a highly conserved tryptophan, the signature residue defining the PHD subclass of Zn fingers, which suggests that other PHD proteins may also be assisted by co-factors in their decoding of modified histone H3 tails.

The interaction of GHRH with membrane-bound receptors on somatotroph cells of the anterior pituitary is an important step in the regulation of GH synthesis and secretion. The identification of a G protein-coupled receptor for GHRH has made it possible to investigate the pathway by which GHRH regulates pituitary somatotroph cell function. To initiate an analysis of the mechanisms regulating expression and function of the GHRH receptor, the structure of the gene and its promoter region were analyzed. The coding sequence of the rat GHRH receptor gene is contained within 14 exons spanning approximately 15 kb of genomic DNA. Four transcription start sites are located within 286 bp upstream of the initiation codon. The 5' flanking region of the GHRH receptor gene acts as a functional promoter in rat pituitary tumor GH3 cells, and basal promoter activity is enhanced in GH3 and COS7 cells by cotransfection of an expression construct encoding the pituitary-specific transcription factor Pit-1. The rat GHRH receptor gene is subject to at least 1 alternative RNA processing event that generates 2 receptor isoforms differing by 41 amino acids within the third intracellular loop (IL) of the protein. The short isoform of the GHRH receptor is predominant in pituitary cells. The MtT/S pituitary tumor cell line was found to express the GHRH receptor, and different populations of these cells produce predominantly the long or short isoforms of the receptor messenger RNA, suggesting that the alternative splicing can be regulated. Functional analysis of the two GHRH receptor isoforms demonstrates that both bind GHRH, but only the short isoform signals through a cAMP-mediated pathway. Neither receptor isoform is able to stimulate calcium mobilization from internal stores after GHRH treatment. Our findings indicate that the pituitary-specific transcription factor Pit-1 is involved in the somatotroph-specific expression of the GHRH receptor gene and that functionally distinct receptor proteins are generated by an alternative RNA processing mechanism.