Peptide conjugation to an in vitro-selected DNA ligand improves enzyme inhibition.

An in vitro selection technique was used to identify a specific high-affinity DNA ligand targeted to human neutrophil elastase (HNE). 1H NMR data and a comparative analysis of the selected sequences suggest that the DNA folds into a G-quartet structure with duplexed ends. The high-affinity binding DNA alone did not inhibit the enzymatic activity of HNE. The DNA was covalently attached to a tetrapeptide, N-methoxysuccinyl-Ala-Ala-Pro-Val, that is a weak competitive inhibitor of HNE. HNE was inhibited by this DNA-peptide conjugate nearly five orders of magnitude more effectively than by the peptide alone. These results demonstrate that in vitro-selected nucleic acids can be used as a vehicle for molecular delivery.

The histone fold: a ubiquitous architectural motif utilized in DNA compaction and protein dimerization.

The histones of all eukaryotes show only a low degree of primary structure homology, but our earlier crystallographic results defined a three-dimensional structural motif, the histone fold, common to all core histones. We now examine the specific architectural patterns within the fold and analyze the nature of the amino acid residues within its functional segments. The histone fold emerges as a fundamental protein dimerization motif while the differentiations of the tips of the histone dimers appear to provide the rules of core octamer assembly and the basis for nucleosome regulation. We present evidence for the occurrence of the fold from archaebacteria to mammals and propose the use of this structural motif to define a distinct family of proteins, the histone fold superfamily. It appears that evolution has conserved the conformation of the fold even through variations in primary structure and among proteins with various functional roles.

Inhibition of human immunodeficiency virus type 1 transcription and replication by DNA sequence-selective plant lignans.

A plant lignan, 3'-O-methyl nordihydroguaiaretic acid (3'-O-methyl NDGA, denoted Malachi 4:5-6 or Mal.4; molecular weigth 316), was isolated from Larrea tridentata and found to be able to inhibit human immunodeficiency virus (HIV) Tat-regulated transactivation in vivo, induce protection of lymphoblastoid CEM-SS cells from HIV (strain IIIB) killing, and suppress the replication of five HIV-1 strains (WM, MN, VS, JR-CSF, and IIIB) in mitogen-stimulated peripheral blood mononuclear cells, all in a dose-dependent manner. Mal.4 inhibits both basal transcription and Tat-regulated transactivation in vitro. The target of Mal.4 has been localized to nucleotides -87 to -40 of the HIV long terminal repeat. Mal.4 directly and specifically interferes with the binding of Sp1 to Sp1 sites in the HIV long terminal repeat. By inhibiting proviral expression, Mal.4 may be able to interrupt the life cycles of both wild-type and reverse transcriptase or protease mutant viruses in HIV-infected patients.

Covalent protein-DNA complexes at the 5' strand termini of meiosis-specific double-strand breaks in yeast.

During meiosis in Saccharomyces cerevisiae, the first chemical step in homologous recombination is the occurrence of site-specific DNA double-strand breaks (DSBs). In wild-type cells, these breaks undergo resection of their 5' strand termini to yield molecules with 3' single-stranded tails. We have further characterized the breaks that accumulate in rad50S mutant stains defective in DSB resection. We find that these DSBs are tightly associated with protein via what appears to be a covalent linkage. When genomic DNA is prepared from meiotic rad50S cultures without protease treatment steps, the restriction fragments diagnostic of DSBs selectively partition to the organic-aqueous interphase in phenol extractions and band at lower than normal density in CsCl density gradients. Selective partitioning and decreased buoyant density are abolished if the DNA is treated with proteinase K prior to analysis. Similar results are obtained with sae2-1 mutant strains, which have phenotypes identical to rad50S mutants. The protein is bound specifically to the 5' strand termini of DSBs and is present at both 5' ends in at least a fraction of breaks. The stability of the complex to various protein denaturants and the strand specificity of the attachment are most consistent with a covalent linkage to DSB termini. We propose that the DSB-associated protein is the catalytic subunit of the meiotic recombination initiation nuclease and that it cleaves DNA via a covalent protein-DNA intermediate.

Inferring phylogenies from DNA sequences of unequal base compositions.

A new method for computing evolutionary distances between DNA sequences is proposed. Contrasting with classical methods, the underlying model does not assume that sequence base compositions (A, C, G, and T contents) are at equilibrium, thus allowing unequal base compositions among compared sequences. This makes the method more efficient than the usual ones in recovering phylogenetic trees from sequence data when base composition is heterogeneous within the data set, as we show by using both simulated and empirical data. When applied to small-subunit ribosomal RNA sequences from several prokaryotic or eukaryotic organisms, this method provides evidence for an early divergence of the microsporidian Vairimorpha necatrix in the eukaryotic lineage.

Calicheamicin-DNA complexes: warhead alignment and saccharide recognition of the minor groove.

The solution structures of calicheamicin gamma 1I, its cycloaromatized analog (calicheamicin epsilon), and its aryl tetrasaccharide complexed to a common DNA hairpin duplex have been determined by NMR and distance-refined molecular dynamics computations. Sequence specificity is associated with carbohydrate-DNA recognition that places the aryl tetrasaccharide component of all three ligands in similar orientations in the minor groove at the d(T-C-C-T).d(A-G-G-A) segment. The complementary fit of the ligands and the DNA minor groove binding site creates numerous van der Waals contacts as well as hydrogen bonding interactions. Notable are the iodine and sulfur atoms of calicheamicin that hydrogen bond with the exposed amino proton of the 5'- and 3'-guanines, respectively, of the d(A-G-G-A) segment. The sequence-specific carbohydrate binding orients the enediyne aglycone of calicheamicin gamma 1I such that its C3 and C6 proradical centers are adjacent to the cleavage sites. While the enediyne aglycone of calicheamicin gamma 1I is tilted relative to the helix axis and spans the minor groove, the cycloaromatized aglycone is aligned approximately parallel to the helix axis in the respective complexes. Specific localized conformational perturbations in the DNA have been identified from imino proton complexation shifts and changes in specific sugar pucker patterns on complex formation. The helical parameters for the carbohydrate binding site are comparable with corresponding values in B-DNA fibers while a widening of the groove is observed at the adjacent aglycone binding site.

A single-stranded DNA binding protein binds the origin of replication of the duplex kinetoplast DNA.

Replication of the kinetoplast DNA (kDNA) minicircle of trypanosomatids initiates at a conserved 12-nt sequence, 5'-GGGGTTGGTGTA-3', termed the universal minicircle sequence (UMS). A sequence-specific single-stranded DNA-binding protein from Crithidia fasciculata binds the heavy strand of the 12-mer UMS. Whereas this UMS-binding protein (UMSBP) does not bind a duplex UMS dodecamer, it binds the double-stranded kDNA minicircle as well as a duplex minicircle fragment containing the origin-associated UMS. Binding of the minicircle origin region by the single-stranded DNA binding protein suggested the local unwinding of the DNA double helix at this site. Modification of thymine residues at this site by KMnO4 revealed that the UMS resides within an unwound or otherwise sharply distorted DNA at the minicircle origin region. Computer analysis predicts the sequence-directed curving of the minicircle origin region. Electrophoresis of a minicircle fragment containing the origin region in polyacrylamide gels revealed a significantly lower electrophoretic mobility than expected from its length. The fragment anomalous electrophoretic mobility is displayed only in its native conformation and is dependent on temperature and gel porosity, indicating the local curving of the DNA double helix. We suggest that binding of UMSBP at the minicircle origin of replication is possible through local unwinding of the DNA double helix at the UMS site. It is hypothesized here that this local melting is initiated through the untwisting of unstacked dinucleotide sequences at the bent origin site.

Isolation, regulation, and DNA-binding properties of three Drosophila nuclear hormone receptor superfamily members.

We have designed a rapid cloning and screening strategy to identify new members of the nuclear hormone receptor superfamily that are expressed during the onset of Drosophila metamorphosis. Using this approach, we isolated three Drosophila genes, designated DHR38, DHR78, and DHR96. All three genes are expressed throughout third-instar larval and prepupal development. DHR38 is the Drosophila homolog of NGFI-B and binds specifically to an NGFI-B response element. DHR78 and DHR96 are orphan receptor genes. DHR78 is induced by 20-hydroxyecdysone (20E) in cultured larval organs, and its encoded protein binds to two AGGTCA half-sites arranged as either direct or palindromic repeats. DHR96 is also 20E-inducible, and its encoded protein binds selectively to the hsp27 20E response element. The 20E receptor can bind to each of the sequences recognized by DHR78 and DHR96, indicating that these proteins may compete with the receptor for binding to a common set of target sequences.

Identification of a DNA element determining synaptic expression of the mouse acetylcholine receptor delta-subunit gene.

mRNAs for acetylcholine receptor genes are highly concentrated in the endplate region of adult skeletal muscle largely as a result of a transcription restricted to the subneural nuclei. To identify the regulatory elements involved, we employed a DNA injection of a plasmid containing a fragment of the acetylcholine receptor delta-subunit gene promoter (positions -839 to +45) linked to the reporter gene lacZ with a nuclear localization signal. Injection of the wild-type construct into mouse leg muscles yielded preferential expression of the reporter gene in the synaptic region. Analysis of various mutant promoters resulted in the identification of a DNA element (positions -60 to -49), referred to as the N box, that plays a critical role in subneural expression. Disruption of this 12-bp element in the context of a mouse delta-subunit promoter from positions -839 to +45 gives widespread expression of the reporter gene throughout the entire muscle fiber, indicating that this element is a silencer that represses delta-subunit gene transcription in extrajunctional areas. On the other hand, this element inserted upstream of a heterologous basal promoter preferentially enhances expression in the endplate region. This element therefore regulates the restricted expression of the delta-subunit gene both as an enhancer at the endplate level and as a silencer in extrajunctional areas. Furthermore, gel-shift experiments with mouse muscle extracts reveal an activity that specifically binds the 6-bp sequence TTCCGG of this element, suggesting that a transcription factor(s) controls the expression of the delta-subunit gene via this element.

Changing nucleosome positions through modification of the DNA rotational information.

The effects of the rotational information of DNA in determining the in vitro localization of nucleosomal core particles (ncps) have been studied in the Saccharomyces cerevisiae 5S rRNA repeat gene. We have altered the distribution of the phased series of flexibility signals present on this DNA by inserting a 25-bp tract, and we have analyzed the effects of this mutation on the distribution and on the frequencies of ncps, as compared with the wild type and a reference 21-bp insertion mutant. The variation of the standard free energy of nucleosome reconstitution was determined. The results show that the DNA rotational information is a major determinant of ncps positioning, define how many rotationally phased signals are required for the formation of a stable particle, and teach how to modify their distribution through the alteration of the rotational signals.

Crystal structure of a DNA decamer showing a novel pseudo four-way helix-helix junction.

The crystal structure of the decanucleotide d(CGCAATTGCG)2 has been solved by a combination of molecular replacement and heavy-atom procedures and has been refined to an R factor of 20.2% at 2.7 A. It is not a fully base-paired duplex but has a central core of eight Watson-Crick base pairs flanked by unpaired terminal guanosines and cytosines. These participate in hydrogen-bonding arrangements with adjacent decamer duplexes in the crystal lattice. The unpaired guanosines are bound in the G+C regions of duplex minor grooves. The cytosines have relatively high mobility, even though they are constrained to be in one region where they are involved in base-paired triplets with G.C base pairs. The 5'-AATT sequence in the duplex region has a narrow minor groove, providing further confirmation of the sequence-dependent nature of groove width.

Gene for the catalytic subunit of mouse DNA-dependent protein kinase maps to the scid locus.

The gene encoding the catalytic subunit of DNA-dependent protein kinase (DNA-PKcs) has been proposed recently as a candidate gene for the mouse severe combined immune deficiency (scid) locus. We have used a partial cDNA clone for human DNA-PKcs to map the mouse homologue using a large interspecific backcross panel. We found that the mouse gene for DNA-PKcs does not recombine with scid, consistent with the hypothesis that scid is a mutation in the mouse gene for DNA-PKcs.

A DNA sequencing strategy that requires only five bases of known terminal sequence for priming.

We have previously reported an enhanced version of sequencing by hybridization (SBH), termed positional SBH (PSBH). PSBH uses partially duplex probes containing single-stranded 3' overhangs, instead of simple single-stranded probes. Stacking interactions between the duplex probe and a single-stranded target allow us to reduce the probe sizes required to 5-base single-stranded overhangs. Here we demonstrate the use of PSBH to capture relatively long single-stranded DNA targets and perform standard solid-state Sanger sequencing on these primer-template complexes without ligation. Our results indicate that only 5 bases of known terminal sequence are required for priming. In addition, the partially duplex probes have the ability to capture their specific target from a mixture of five single-stranded targets with different 3'-terminal sequences. This indicates the potential utility of the PSBH approach to sequence mixtures of DNA targets without prior purification.

Chloroplast DNA variation and the recent radiation of the giant senecios (Asteraceae) on the tall mountains of eastern Africa.

Chloroplast DNA restriction-site variation was surveyed among 40 accessions representing all 11 species of giant senecios (Dendrosenecio, Asteraceae) at all but one known location, plus three outgroup species. Remarkably little variation (only 9 variable sites out of roughly 1000 sites examined) was found among the 40 giant senecio accessions, yet as a group they differ significantly (at 18 sites) from Cineraria deltoidea, the closest known relative. This pattern indicates that the giant senecios underwent a recent dramatic radiation in eastern Africa and evolved from a relatively isolated lineage within the Senecioneae. Biogeographic interpretation of the molecular phylogeny suggests that the giant senecios originated high on Mt. Kilimanjaro, with subsequent dispersion to the Aberdares, Mt. Kenya, and the Cherangani Hills, followed by dispersion westward to the Ruwenzori Mountains, and then south to the Virunga Mountains, Mt. Kahuzi, and Mt. Muhi, but with dispersion back to Mt. Elgon. Geographic radiation was an important antecedent to the diversification in eastern Africa, which primarily involved repeated altitudinal radiation, both up and down the mountains, leading to morphological parallelism in both directions. In general, the plants on a given mountain are more closely related to each other than they are to plants on other mountains, and plants on nearby mountains are more closely related to each other than they are to plants on more distant mountains. The individual steps of the geographic radiation have occurred at various altitudes, some clearly the result of intermountain dispersal. The molecular evidence suggests that two species are extant ancestors to other species on the same or nearby mountains.

Cyclic polyamides for recognition in the minor groove of DNA.

Small molecules that specifically bind with high affinity to any designated DNA sequence in the human genome would be useful tools in molecular biology and potentially in human medicine. Simple rules have been developed to rationally alter the sequence specificity of minor groove-binding polyamides containing N-methylimidazole and N-methylpyrrole amino acids. Crescent-shaped polyamides bind as antiparallel dimers with each polyamide making specific contacts with each strand on the floor of the minor groove. Cyclic polyamides have now been synthesized that bind designated DNA sequences at subnanomolar concentrations.