Helicase binding to DnaI exposes a cryptic DNA-binding site during helicase loading in Bacillus subtilis

The Bacillus subtilis DnaI, DnaB and DnaD proteins load the replicative ring helicase DnaC onto DNA during priming of DNA replication. Here we show that DnaI consists of a C-terminal domain (Cd) with ATPase and DNA-binding activities and an N-terminal domain (Nd) that interacts with the replicative ring helicase. A Zn²⁺-binding module mediates the interaction with the helicase and C67, C70 and H84 are involved in the coordination of the Zn²⁺. DnaI binds ATP and exhibits ATPase activity that is not stimulated by ssDNA, because the DNA-binding site on Cd is masked by Nd. The ATPase activity resides on the Cd domain and when detached from the Nd domain, it becomes sensitive to stimulation by ssDNA because its cryptic DNA-binding site is exposed. Therefore, Nd acts as a molecular ‘switch’ regulating access to the ssDNA binding site on Cd, in response to binding of the helicase. DnaI is sufficient to load the replicative helicase from a complex with six DnaI molecules, so there is no requirement for a dual helicase loader system.     

The influence of recombination on human genetic diversity

In humans, the rate of recombination, as measured on the megabase scale, is positively associated with the level of genetic variation, as measured at the genic scale. Despite considerable debate, it is not clear whether these factors are causally linked or, if they are, whether this is driven by the repeated action of adaptive evolution or molecular processes such as double-strand break formation and mismatch repair. We introduce three innovations to the analysis of recombination and diversity: fine-scale genetic maps estimated from genotype experiments that identify recombination hotspots at the kilobase scale, analysis of an entire human chromosome, and the use of wavelet techniques to identify correlations acting at different scales. We show that recombination influences genetic diversity only at the level of recombination hotspots. Hotspots are also associated with local increases in GC content and the relative frequency of GC-increasing mutations but have no effect on substitution rates. Broad-scale association between recombination and diversity is explained through covariance of both factors with base composition. To our knowledge, these results are the first evidence of a direct and local influence of recombination hotspots on genetic variation and the fate of individual mutations. However, that hotspots have no influence on substitution rates suggests that they are too ephemeral on an evolutionary time scale to have a strong influence on broader scale patterns of base composition and long-term molecular evolution.     

In the Bacillus stearothermophilus DnaB-DnaG complex, the activities of the two proteins are modulated by distinct but overlapping networks of residues

We demonstrate the primase activity of Bacillus stearothermophilus DnaG and show that it initiates at 3'-ATC-5' and 3'-ATT-5' sites synthesizing primers that are 22 or 23 nucleotides long. In the presence of the helicase DnaB the size distribution of primers is different, and a range of additional smaller primers are also synthesized. Nine residues from the N- and C-terminal domains of DnaB, as well as its linker region, have been reported previously to affect this interaction. In Bacillus stearothermophilus only three residues from the linker region (I119 and I125) and the N-terminal domain (Y88) of DnaB have been shown previously to have direct structural importance, and I119 and I125 mediate DnaG-induced effects on DnaB activity. The functions of the other residues (L138, T191, E192, R195, and M196) are still a mystery. Here we show that the E15A, Y88A, and E15A Y88A mutants bind DnaG but are not able to modulate primer size, whereas the R195A M196A mutant inhibited the primase activity. Therefore, four of these residues, E15 and Y88 (N-terminal domain) and R195 and M196 (C-terminal domain), mediate DnaB-induced effects on DnaG activity. Overall, the data suggest that the effects of DnaB on DnaG activity and vice versa are mediated by distinct but overlapping networks of residues.     

The Human Endometrium Expresses the Glycoprotein Mucin-1 and Shows Positive Correlation for Thomsen-Friedenreich Epitope Expression and Galectin-1 Binding

Mucin 1 (MUC1) is a glycoprotein in human endometrium and is abundant at the luminal epithelial surface in the receptive phase. It has a highly glycosylated ecto-domain that contains keratan sulfate chains, that disappears at the time of implantation. In addition, the glycoforms on MUC1 differ in fertile and infertile women. Therefore the aims of this study were investigations on glycosylation of MUC1 with the Thomsen-Friedenreich (TF) epitope on normal human endometrium throughout the menstrual cycle and binding of galectin-1 on the TF epitope in the endometrium and the expression of galectin-1 on the human oocyte. Human endometrial tissue was obtained from 54 premenopausal patients and was immunohistochemically analyzed with monoclonal antibodies against MUC1, TF epitope, galectin-1, and biotinylated galectin-1. In addition, human oocytes were analyzed for TF, galectin-1 expression, and galectin-1 binding. We identified a significant upregulation of MUC1 and TF epitope and, in addition, galectin-1 binding in glandular epithelium and epithelial apical surface tissue from proliferative to secretory phase. With double staining experiments, we identified a coexpression of TF and MUC1 in the early secretory phase and galectin-1 binding to TF during the same period of time. In addition we identified TF epitope and galectin-1 expression plus binding on the human oocyte and irregularly fertilized oocytes. Upregulation of TF epitope on the glandular epithelium and epithelial apical surface tissue in the secretory phase and binding of galectin-1 at the same time show the possibility of galectin-1–mediated trophectoderm binding to the endometrium within the window of implantation. (J Histochem Cytochem 57:871–881, 2009)     

Integrated genetic and epigenetic analysis identifies haplotype-specific methylation in the FTO type 2 diabetes and obesity susceptibility locus

Recent multi-dimensional approaches to the study of complex disease have revealed powerful insights into how genetic and epigenetic factors may underlie their aetiopathogenesis. We examined genotype-epigenotype interactions in the context of Type 2 Diabetes (T2D), focussing on known regions of genomic susceptibility. We assayed DNA methylation in 60 females, stratified according to disease susceptibility haplotype using previously identified association loci. CpG methylation was assessed using methylated DNA immunoprecipitation on a targeted array (MeDIP-chip) and absolute methylation values were estimated using a Bayesian algorithm (BATMAN). Absolute methylation levels were quantified across LD blocks, and we identified increased DNA methylation on the FTO obesity susceptibility haplotype, tagged by the rs8050136 risk allele A (p = 9.40×10⁻⁴, permutation p = 1.0×10⁻³). Further analysis across the 46 kb LD block using sliding windows localised the most significant difference to be within a 7.7 kb region (p = 1.13×10⁻⁷). Sequence level analysis, followed by pyrosequencing validation, revealed that the methylation difference was driven by the co-ordinated phase of CpG-creating SNPs across the risk haplotype. This 7.7 kb region of haplotype-specific methylation (HSM), encapsulates a Highly Conserved Non-Coding Element (HCNE) that has previously been validated as a long-range enhancer, supported by the histone H3K4me1 enhancer signature. This study demonstrates that integration of Genome-Wide Association (GWA) SNP and epigenomic DNA methylation data can identify potential novel genotype-epigenotype interactions within disease-associated loci, thus providing a novel route to aid unravelling common complex diseases.     

When simple sequence comparison fails: the cryptic case of the shared domains of the bacterial replication initiation proteins DnaB and DnaD

DnaD and DnaB are essential DNA-replication-initiation proteins in low-G+C content Gram-positive bacteria. Here we use sensitive Hidden Markov Model-based techniques to show that the DnaB and DnaD proteins share a common structure that is evident across all their structural domains, termed DDBH1 and DDBH2 (DnaD DnaB Homology 1 and 2). Despite strong sequence divergence, many of the DNA-binding and oligomerization properties of these domains have been conserved. Although eluding simple sequence comparisons, the DDBH2 domains share the only strong sequence motif; an extremely highly conserved YxxxIxxxW sequence that contributes to DNA binding. Sequence alignments of DnaD alone fail to identify another key part of the DNA-binding module, since it includes a poorly conserved sequence, a solvent-exposed and somewhat unstable helix and a mobile segment. We show by NMR, in vitro mutagenesis and in vivo complementation experiments that the DNA-binding module of Bacillus subtilis DnaD comprises the YxxxIxxxW motif, the unstable helix and a portion of the mobile region, the latter two being essential for viability. These structural insights lead us to a re-evaluation of the oligomerization and DNA-binding properties of the DnaD and DnaB proteins.     

Pharmacological Screening Using an FXN-EGFP Cellular Genomic Reporter Assay for the Therapy of Friedreich Ataxia

Friedreich ataxia (FRDA) is an autosomal recessive disorder characterized by neurodegeneration and cardiomyopathy. The presence of a GAA trinucleotide repeat expansion in the first intron of the FXN gene results in the inhibition of gene expression and an insufficiency of the mitochondrial protein frataxin. There is a correlation between expansion length, the amount of residual frataxin and the severity of disease. As the coding sequence is unaltered, pharmacological up-regulation of FXN expression may restore frataxin to therapeutic levels. To facilitate screening of compounds that modulate FXN expression in a physiologically relevant manner, we established a cellular genomic reporter assay consisting of a stable human cell line containing an FXN-EGFP fusion construct, in which the EGFP gene is fused in-frame with the entire normal human FXN gene present on a BAC clone. The cell line was used to establish a fluorometric cellular assay for use in high throughput screening (HTS) procedures. A small chemical library containing FDA-approved compounds and natural extracts was screened and analyzed. Compound hits identified by HTS were further evaluated by flow cytometry in the cellular genomic reporter assay. The effects on FXN mRNA and frataxin protein levels were measured in lymphoblast and fibroblast cell lines derived from individuals with FRDA and in a humanized GAA repeat expansion mouse model of FRDA. Compounds that were established to increase FXN gene expression and frataxin levels included several anti-cancer agents, the iron-chelator deferiprone and the phytoalexin resveratrol.     

Phosphorylation-mediated 14-3-3 Protein Binding Regulates the Function of the Rho-specific Guanine Nucleotide Exchange Factor (RhoGEF) Syx

Syx is a Rho-specific guanine nucleotide exchange factor (GEF) that localizes at cell-cell junctions and promotes junction stability by activating RhoA and the downstream effector Diaphanous homolog 1 (Dia1). Previously, we identified several molecules, including 14-3-3 proteins, as Syx-interacting partners. In the present study, we show that 14-3-3 isoforms interact with Syx at both its N- and C-terminal regions in a phosphorylation-dependent manner. We identify the protein kinase D-mediated phosphorylation of serine 92 on Syx, and additional phosphorylation at serine 938, as critical sites for 14-3-3 association. Our data indicate that the binding of 14-3-3 proteins inhibits the GEF activity of Syx. Furthermore, we show that phosphorylation-deficient, 14-3-3-uncoupled Syx exhibits increased junctional targeting and increased GEF activity, resulting in the strengthening of the circumferential junctional actin ring in Madin-Darby canine kidney cells. These findings reveal a novel means of regulating junctional Syx localization and function by phosphorylation-induced 14-3-3 binding and further support the importance of Syx function in maintaining stable cell-cell contacts.