DNA binding sites for the Mlc and NagC proteins: regulation of nagE, encoding the N-acetylglucosamine-specific transporter in Escherichia coli

The NagC and Mlc proteins are homologous transcriptional regulators that control the expression of several phosphotransferase system (PTS) genes in Escherichia coli. NagC represses nagE, encoding the N-acetylglucosamine-specific transporter, while Mlc represses three PTS operons, ptsG, manXYZ and ptsHIcrr, involved in the uptake of glucose. NagC and Mlc can bind to each others operator, at least in vitro. A binding site selection procedure was used to try to distinguish NagC and Mlc sites. The major difference was that all selected NagC binding sites had a G or a C at positions +11/–11 from the centre of symmetry. This is also the case for most native NagC sites, but not the nagE operator, which thus looks like a potential Mlc target. The nagE operator does exhibit a higher affinity for Mlc than NagC, but no regulation of nagE by physiological concentrations of Mlc was detected in vivo. Regulation of wild-type nagE by NagC is achieved because of the chelation effect due to a second high affinity NagC operator covering the nagB promoter. Replacing the A/T at +11/–11 with C/G allows repression by NagC in the absence of the nagB operator.

DNA-binding and strand-annealing activities of human Mre11: implications for its roles in DNA double-strand break repair pathways

DNA double-strand breaks (DSBs) in eukaryotic cells can be repaired by non-homologous end-joining or homologous recombination. The complex containing the Mre11, Rad50 and Nbs1 proteins has been implicated in both DSB repair pathways, even though they are mechanistically different. To get a better understanding of the properties of the human Mre11 (hMre11) protein, we investigated some of its biochemical activities. We found that hMre11 binds both double- and single-stranded (ss)DNA, with a preference for ssDNA. hMre11 does not require DNA ends for efficient binding. Interestingly, hMre11 mediates the annealing of complementary ssDNA molecules. In contrast to the annealing activity of the homologous recombination protein hRad52, the activity of hMre11 is abrogated by the ssDNA binding protein hRPA. We discuss the possible implications of the results for the role(s) of hMre11 in both DSB repair pathways.

The Homeodomain Resource: sequences, structures, DNA binding sites and genomic information

The Homeodomain Resource is an annotated collection of non-redundant protein sequences, three-dimensional structures and genomic information for the homeodomain protein family. Release 3.0 contains 795 full-length homeodomain-containing sequences, 32 experimentally-derived structures and 143 homeo­box loci implicated in human genetic disorders. Entries are fully hyperlinked to facilitate easy retrieval of the original records from source databases. A simple search engine with a graphical user interface is provided to query the component databases and assemble customized data sets. A new feature for this release is the addition of DNA recognition sites for all human homeodomain proteins described in the literature. The Homeodomain Resource is freely available through the World Wide Web at http://genome.nhgri.nih.gov/homeodomain.

Specific down-regulation of an engineered human cyclin D1 promoter by a novel DNA-binding ligand in intact cells

Cyclin D1 is expressed at abnormally high levels in many cancers and has been specifically implicated in the development of breast cancer. In this report we have extensively analyzed the cyclin D1 promoter in a variety of cancer cell lines that overexpress the protein and identified two critical regulatory elements (CREs), a previously identified CRE at –52 and a novel site at –30. In vivo footprinting experiments demonstrated factors binding at both sites. We have used a novel DNA-binding ligand, GL020924, to target the site at –30 (–30–21) of the cyclin D1 promoter in MCF7 breast cancer cells. A binding site for this novel molecule was constructed by mutating 2 bp of the wild-type cyclin D1 promoter at the –30–21 site. Treatment with GL020924 specifically inhibited expression of the targeted cyclin D1 promoter construct in MCF7 cells in a concentration-dependent manner, thus validating the –30–21 site as a target for minor groove-binding ligands. In addition, this result validates our approach to regulating the expression of genes implicated in disease by targeting small DNA-binding ligands to key regulatory elements in the promoters of those genes.

An initial ATP-independent step in the unwinding of a herpes simplex virus type I origin of replication by a complex of the viral origin-binding protein and single-strand DNA-binding protein

Using a spectrophotometric assay that measures the hyperchromicity that accompanies the unwinding of a DNA duplex, we have identified an ATP-independent step in the unwinding of a herpes simplex virus type 1 (HSV-1) origin of replication, Oris, by a complex of the HSV-1 origin binding protein (UL9 protein) and the HSV-1 single-strand DNA binding protein (ICP8). The sequence unwound is the 18-bp A + T-rich segment that links the two high-affinity UL9 protein binding sites, boxes I and II of Oris. P1 nuclease sensitivity of Oris and single-strand DNA-dependent ATPase measurements of the UL9 protein indicate that, at 37°C, the A + T-rich segment is sufficiently single stranded to permit the binding of ICP8. Binding of the UL9 protein to boxes I and II then results in the formation of the UL9 protein–ICP8 complex, that can, in the absence of ATP, promote unwinding of the A + T-rich segment. On addition of ATP, the helicase activity of the UL9 protein–ICP8 complex can unwind boxes I and II, permitting access of the replication machinery to the Oris sequences.

Crystal structure of the C-terminal domain of the RAP74 subunit of human transcription factor IIF

The x-ray structure of a C-terminal fragment of the RAP74 subunit of human transcription factor (TF) IIF has been determined at 1.02-Å resolution. The α/β structure is strikingly similar to the globular domain of linker histone H5 and the DNA-binding domain of hepatocyte nuclear factor 3γ (HNF-3γ), making it a winged-helix protein. The surface electrostatic properties of this compact domain differ significantly from those of bona fide winged-helix transcription factors (HNF-3γ and RFX1) and from the winged-helix domains found within the RAP30 subunit of TFIIF and the β subunit of TFIIE. RAP74 has been shown to interact with the TFIIF-associated C-terminal domain phosphatase FCP1, and a putative phosphatase binding site has been identified within the RAP74 winged-helix domain.

Activation of Trp3 by inositol 1,4,5-trisphosphate receptors through displacement of inhibitory calmodulin from a common binding domain

Mammalian homologues of Drosophila Trp form plasma membrane channels that mediate Ca2+ influx in response to activation of phospholipase C and internal Ca2+ store depletion. Previous studies showed that human Trp3 is activated by inositol 1,4,5-trisphosphate (IP3) receptors (IP3Rs) and identified interacting domains, one on Trp and two on IP3R. We now find that Trp3 binds Ca2+-calmodulin (Ca2+/CaM) at a site that overlaps with the IP3R binding domain. Using patch-clamp recordings from inside-out patches, we further show that Trp3 has a high intrinsic activity that is suppressed by Ca2+/CaM under resting conditions, and that Trp3 is activated by the following: a Trp-binding peptide from IP3R that displaces CaM from Trp3, a myosin light chain kinase Ca2+/CaM binding peptide that prevents CaM from binding to Trp3, and calmidazolium, an inactivator of Ca2+/CaM. We conclude that inhibition of the inhibitory action of CaM is a key step of Trp3 channel activation by IP3Rs.

ADP-Dependent Phosphorylation Regulates Association of a DNA-Binding Complex with the Barley Chloroplast psbD Blue-Light-Responsive Promoter1

The chloroplast gene psbD encodes D2, a chlorophyll-binding protein located in the photosystem II reaction center. Transcription of psbD in higher plants involves at least three promoters, one of which is regulated by blue light. The psbD blue-light-regulated promoter (BLRP) consists of a −10 promoter element and an activating complex, AGF, that binds immediately upstream of −35. A second sequence-specific DNA-binding complex, PGTF, binds upstream of AGF between −71 and −100 in the barley (Hordeum vulgare) psbD BLRP. In this study we report that ADP-dependent phosphorylation selectively inhibits the binding of PGTF to the barley psbD BLRP. ATP at high concentrations (1–5 mm) inhibits PGTF binding, but in the presence of phosphocreatine and phosphocreatine kinase, this capacity is lost, presumably due to scavenging of ADP. ADP inhibits PGTF binding at relatively low concentrations (0.1 mm), whereas other nucleotides are unable to mediate this response. ADP-mediated inhibition of PGTF binding is reduced in the presence of the protein kinase inhibitor K252a. This and other results suggest that ADP-dependent phosphorylation of PGTF (or some associated protein) inhibits binding of PGTF to the psbD BLRP and reduces transcription. ADP-dependent phosphorylation is expected to increase in darkness in parallel with the rise in ADP levels in chloroplasts. ADP-dependent phosphorylation in chloroplasts may, therefore, in coordination, inactivate enzymes involved in carbon assimilation, protein synthesis, and transcription during diurnal light/dark cycles.

Mitochondrial Single-stranded DNA-binding Protein Is Required for Mitochondrial DNA Replication and Development in Drosophila melanogaster

The discovery that several inherited human diseases are caused by mtDNA depletion has led to an increased interest in the replication and maintenance of mtDNA. We have isolated a new mutant in the lopo (low power) gene from Drosophila melanogaster affecting the mitochondrial single-stranded DNA-binding protein (mtSSB), which is one of the key components in mtDNA replication and maintenance. lopo1 mutants die late in the third instar before completion of metamorphosis because of a failure in cell proliferation. Molecular, histochemical, and physiological experiments show a drastic decrease in mtDNA content that is coupled with the loss of respiration in these mutants. However, the number and morphology of mitochondria are not greatly affected. Immunocytochemical analysis shows that mtSSB is expressed in all tissues but is highly enriched in proliferating tissues and in the developing oocyte. lopo1 is the first mtSSB mutant in higher eukaryotes, and its analysis demonstrates the essential function of this gene in development, providing an excellent model to study mitochondrial biogenesis in animals.

Strong DNA binding by covalently linked dimeric Lac headpiece: Evidence for the crucial role of the hinge helices

The combined structural and biochemical studies on Lac repressor bound to operator DNA have demonstrated the central role of the hinge helices in operator bending and the induction mechanism. We have constructed a covalently linked dimeric Lac-headpiece that binds DNA with four orders of magnitude higher affinity as compared with the monomeric form. This enabled a detailed biochemical and structural study of Lac binding to its cognate wild-type and selected DNA operators. The results indicate a profound contribution of hinge helices to the stability of the protein–DNA complex and highlight their central role in operator recognition. Furthermore, protein–DNA interactions in the minor groove appear to modulate hinge helix stability, thus accounting for affinity differences and protein-induced DNA bending among the various operator sites. Interestingly, the in vitro DNA-binding affinity of the reported dimeric Lac construct can de readily modulated by simple adjustment of redox conditions, thus rendering it a potential artificial gene regulator.

Bacterial Cellulose-Binding Domain Modulates in Vitro Elongation of Different Plant Cells1

Recombinant cellulose-binding domain (CBD) derived from the cellulolytic bacterium Clostridium cellulovorans was found to modulate the elongation of different plant cells in vitro. In peach (Prunus persica L.) pollen tubes, maximum elongation was observed at 50 μg mL−1 CBD. Pollen tube staining with calcofluor showed a loss of crystallinity in the tip zone of CBD-treated pollen tubes. At low concentrations CBD enhanced elongation of Arabidopsis roots. At high concentrations CBD dramatically inhibited root elongation in a dose-responsive manner. Maximum effect on root hair elongation was at 100 μg mL−1, whereas root elongation was inhibited at that concentration. CBD was found to compete with xyloglucan for binding to cellulose when CBD was added first to the cellulose, before the addition of xyloglucan. When Acetobacter xylinum L. was used as a model system, CBD was found to increase the rate of cellulose synthase in a dose-responsive manner, up to 5-fold compared with the control. Electron microscopy examination of the cellulose ribbons produced by A. xylinum showed that CBD treatment resulted in a splayed ribbon composed of separate fibrillar subunits, compared with a thin, uniform ribbon in the control.

Characterization of DNA-Binding Proteins from Pea Mitochondria1

We studied transcription initiation in the mitochondria of higher plants, with particular respect to promoter structures. Conserved elements of these promoters have been successfully identified by in vitro transcription systems in different species, whereas the involved protein components are still unknown. Proteins binding to double-stranded oligonucleotides representing different parts of the pea (Pisum sativum) mitochondrial atp9 were analyzed by denaturation-renaturation chromatography and mobility-shift experiments. Two DNA-protein complexes were detected, which appeared to be sequence specific in competition experiments. Purification by hydroxyapatite, phosphocellulose, and reversed-phase high-pressure liquid chromatography separated two polypeptides with apparent molecular masses of 32 and 44 kD. Both proteins bound to conserved structures of the pea atp9 and the heterologous Oenothera berteriana atp1 promoters and to sequences just upstream. Possible functions of these proteins in mitochondrial promoter recognition are discussed.

Defining the roles of individual residues in the single-stranded DNA binding site of PcrA helicase

Crystal structures and biochemical analyses of PcrA helicase provide evidence for a model for processive DNA unwinding that involves coupling of single-stranded DNA (ssDNA) tracking to a duplex destabilization activity. The DNA tracking model invokes ATP-dependent flipping of bases between several pockets on the enzyme formed by conserved aromatic amino acid residues. We have used site-directed mutagenesis to confirm the requirement of all of these residues for helicase activity. We also demonstrate that the duplex unwinding defects correlate with an inability of certain mutant proteins to translocate effectively on ssDNA. Moreover, the results define an essential triad of residues within the ssDNA binding site that comprise the ATP-driven DNA motor itself.

Histone deacetylase-dependent transcriptional repression by pRB in yeast occurs independently of interaction through the LXCXE binding cleft

We have developed a yeast model system to address transcriptional repression by the retinoblastoma protein (pRB). When fused to the DNA-binding domain of Gal4p (DB-pRB), pRB can repress transcription of reporter genes containing Gal4p binding sites; the histone deacetylase activity encoded by yeast RPD3 is required for DB-pRB repression. Mutation of the LXCXE binding cleft in pRB, a region reported to be required for histone deacetylase recruitment, does not interfere with pRB-mediated repression. From these findings based on yeast experiments, we surmise that the small pocket region of pRB must contain an additional domain that confers histone deacetylase-dependent transcriptional repression. This hypothesis was verified by experiments examining pRB-dependent histone deacetylase association in mammalian cells. In addition to RPD3, repression by pRB in yeast requires MSI1, an ortholog of RbAp48, but not SIN3 or SAP30. By comparing the genetic requirements of DB-pRB repression in yeast to those of other DB-repressor fusions, we can suggest a mechanism by which pRB recruits histone deacetylase activity.