Phosphorylated and unphosphorylated forms of human single-stranded DNA-binding protein are equally active in simian virus 40 DNA replication and in nucleotide excision repair.

The trimeric human single-stranded DNA-binding protein (HSSB; also called RP-A) plays an essential role in DNA replication, nucleotide excision repair, and homologous DNA recombination. The p34 subunit of HSSB is phosphorylated at the G1/S boundary of the cell cycle or upon exposure of cells to DNA damage-inducing agents including ionizing and UV radiation. We have previously shown that the phosphorylation of p34 is catalyzed by both cyclin-dependent kinase-cyclin A complex and DNA-dependent protein kinase. In this study, we investigated the effect of phosphorylation of p34 by these kinases on the replication and repair function of HSSB. We observed no significant difference with the unphosphorylated and phosphorylated forms of HSSB in the simian virus 40 DNA replication or nucleotide excision repair systems reconstituted with purified proteins. The phosphorylation status of the p34 subunit of HSSB was unchanged during the reactions. We suggest that the phosphorylated HSSB has no direct effect on the basic mechanism of DNA replication and nucleotide excision repair reactions in vitro, although we cannot exclude a role of p34 phosphorylation in modulating HSSB function in vivo through a yet poorly understood control pathway in the cellular response to DNA damage and replication.

Bacteriophage T7 helicase/primase proteins form rings around single-stranded DNA that suggest a general structure for hexameric helicases.

Most helicases studied to date have been characterized as oligomeric, but the relation between their structure and function has not been understood. The bacteriophage T7 gene 4 helicase/primase proteins act in T7 DNA replication. We have used electron microscopy, three-dimensional reconstruction, and protein crosslinking to demonstrate that both proteins form hexameric rings around single-stranded DNA. Each subunit has two lobes, so the hexamer appears to be two-tiered, with a small ring stacked on a large ring. The single-stranded DNA passes through the central hole of the hexamer, and the data exclude substantial wrapping of the DNA about or within the protein ring. Further, the hexamer binds DNA with a defined polarity as the smaller ring of the hexamer points toward the 5' end of the DNA. The similarity in three-dimensional structure of the T7 gene 4 proteins to that of the Escherichia coli RuvB helicase suggests that polar rings assembled around DNA may be a general feature of numerous hexameric helicases involved in DNA replication, transcription, recombination, and repair.

Y box-binding protein-1 binds preferentially to single-stranded nucleic acids and exhibits 3′→5′ exonuclease activity

We have previously shown that Y box-binding protein-1 (YB-1) binds preferentially to cisplatin-modified Y box sequences. Based on structural and biochemical data, we predicted that this protein binds single-stranded nucleic acids. In the present study we confirmed the prediction and also discovered some unexpected functional features of YB-1. We found that the cold shock domain of the protein is necessary but not sufficient for double-stranded DNA binding while the C-tail domain interacts with both single-stranded DNA and RNA independently of the cold shock domain. In an in vitro translation system the C-tail domain of the protein inhibited translation but the cold shock domain did not. Both in vitro pull-down and in vivo co-immunoprecipitation assays revealed that YB-1 can form a homodimer. Deletion analysis mapped the C-tail domain of the protein as the region of homodimerization. We also characterized an intrinsic 3′→5′ DNA exonuclease activity of the protein. The region between residues 51 and 205 of its 324-amino acid extent is required for full exonuclease activity. Our findings suggest that YB-1 functions in regulating DNA/RNA transactions and that these actions involve different domains.

RecA.oligonucleotide filaments bind in the minor groove of double-stranded DNA.

Escherichia coli RecA protein, in the presence of ATP or its analog adenosine 5'-[gamma-thio]triphosphate, polymerizes on single-stranded DNA to form nucleoprotein filaments that can then bind to homologous sequences on duplex DNA. The three-stranded joint molecule formed as a result of this binding event is a key intermediate in general recombination. We have used affinity cleavage to examine this three-stranded joint by incorporating a single thymidine-EDTA.Fe (T*) into the oligonucleotide part of the filament. Our analysis of the cleavage patterns from the joint molecule reveals that the nucleoprotein filament binds in the minor groove of an extended Watson-Crick duplex.

RecA binding to a single double-stranded DNA molecule: A possible role of DNA conformational fluctuations

Most genetic regulatory mechanisms involve protein–DNA interactions. In these processes, the classical Watson–Crick DNA structure sometimes is distorted severely, which in turn enables the precise recognition of the specific sites by the protein. Despite its key importance, very little is known about such deformation processes. To address this general question, we have studied a model system, namely, RecA binding to double-stranded DNA. Results from micromanipulation experiments indicate that RecA binds strongly to stretched DNA; based on this observation, we propose that spontaneous thermal stretching fluctuations may play a role in the binding of RecA to DNA. This has fundamental implications for the protein–DNA binding mechanism, which must therefore rely in part on a combination of flexibility and thermal fluctuations of the DNA structure. We also show that this mechanism is sequence sensitive. Theoretical simulations support this interpretation of our experimental results, and it is argued that this is of broad relevance to DNA–protein interactions.

High efficiency mutagenesis, repair, and engineering of chromosomal DNA using single-stranded oligonucleotides

Homologous DNA recombination is a fundamental, regenerative process within living organisms. However, in most organisms, homologous recombination is a rare event, requiring a complex set of reactions and extensive homology. We demonstrate in this paper that Beta protein of phage λ generates recombinants in chromosomal DNA by using synthetic single-stranded DNAs (ssDNA) as short as 30 bases long. This ssDNA recombination can be used to mutagenize or repair the chromosome with efficiencies that generate up to 6% recombinants among treated cells. Mechanistically, it appears that Beta protein, a Rad52-like protein, binds and anneals the ssDNA donor to a complementary single-strand near the DNA replication fork to generate the recombinant. This type of homologous recombination with ssDNA provides new avenues for studying and modifying genomes ranging from bacterial pathogens to eukaryotes. Beta protein and ssDNA may prove generally applicable for repairing DNA in many organisms.

In vivo expression of a single viral DNA-binding protein generates systemic lupus erythematosus-related autoimmunity to double-stranded DNA and histones.

Although the origin of autoimmune antibodies to double-stranded DNA is not known, the variable-region structures of such antibodies indicate that they are produced in response to antigen-selective stimulation. In accordance with this, results from experiments using artificial complexes of DNA and DNA-binding polypeptides for immunizations have indicated that DNA may induce these antibodies. Hence, the immunogenicity of DNA in vivo may depend upon other structures or processes that may render DNA immunogenic. We report that in vivo expression of a single DNA-binding protein, the polyoma virus T antigen, is sufficient to initiate production of anti-double-stranded DNA and anti-histone antibodies but not a panel of other autoantigens. Expression of a mutant, non-DNA-binding T antigen did result in strong production of antibodies to the T antigen, but only borderline levels of antibodies to DNA and no detectable antibodies to histones. Nonexpressing plasmid DNA containing the complete cDNA sequence for T antigen did not evoke such immune responses, indicating that DNA by itself is not immunogenic in vivo. The results represent a conceptual advance in understanding a potential molecular basis for initiation of autoimmunity in systemic lupus erythematosus.

A class of single-stranded telomeric DNA-binding proteins required for Rap1p localization in yeast nuclei.

We have identified a class of proteins that bind single-stranded telomeric DNA and are required for the nuclear organization of telomeres and/or telomere-associated proteins. Rlf6p was identified by its sequence similarity to Gbp1p, a single-stranded telomeric DNA-binding protein from Chlamydomonas reinhardtii. Rlf6p and Gbp1p bind yeast single-stranded G-strand telomeric DNA. Both proteins include at least two RNA recognition motifs, which are found in many proteins that interact with single-stranded nucleic acids. Disruption of RLF6 alters the distribution of repressor/activator protein 1 (Rap1p), a telomere-associated protein. In wild-type yeast cells, Rap1p localizes to a small number of perinuclear spots, while in rlf6 cells Rap1p appears diffuse and nuclear. Interestingly, telomere position effect and telomere length control, which require RAP1, are unaffected by rlf6 mutations, demonstrating that Rap1p localization can be uncoupled from other Rap1p-dependent telomere functions. In addition, expression of Chlamydomonas GBP1 restores perinuclear, punctate Rap1p localization in rlf6 mutant cells. The functional complementation of a fungal gene by an algal gene suggests that Rlf6p and Gbp1p are members of a conserved class of single-stranded telomeric DNA-binding proteins that influence nuclear organization. Furthermore, it demonstrates that, despite their unusual codon bias, C. reinhardtii genes can be efficiently translated in Saccharomyces cerevisiae cells.

The single minichromosome maintenance protein of Methanobacterium thermoautotrophicum ΔH contains DNA helicase activity

Previous studies have identified an ATP-dependent DNA helicase activity intrinsic to the human minichromosome maintenance (MCM) complex, composed of MCM subunits 4, 6, and 7 [Ishimi, Y. (1997) J. Biol. Chem. 272, 24508–24513]. In contrast to the presence of multiple MCM genes (at least six) in eukaryotes, the archaeon Methanobacterium thermoautotrophicum ΔH (mth) genome contains a single open reading frame coding for an MCM protein. In this study we report the isolation of the mthMCM protein overexpressed in Escherichia coli. The purified recombinant protein was found to exist in both multimeric (≈103 kDa) and monomeric (76 kDa) forms. Both forms of the protein bind to single-stranded DNA, hydrolyze ATP in the presence of DNA, and possess 3′-to-5′ ATP-dependent DNA helicase activities. Thus, a single mthMCM protein contains biochemical properties identical to those associated with the eukaryotic MCM4, -6, and -7 complex. These results suggest that the characterization of the mthMCM protein and its multiple forms may contribute to our understanding of the role of MCM helicase activity in eukaryotic chromosomal DNA replication.

Protein-free parallel triple-stranded DNA complex formation

A 14 nt DNA sequence 5′-AGAATGTGGCAAAG-3′ from the zinc finger repeat of the human KRAB zinc finger protein gene ZNF91 bearing the intercalator 2-methoxy,6-chloro,9-amino acridine (Acr) attached to the sugar–phosphate backbone in various positions has been shown to form a specific triple helix (triplex) with a 16 bp hairpin (intramolecular) or a two-stranded (intermolecular) duplex having the identical sequence in the same (parallel) orientation. Intramolecular targets with the identical sequence in the antiparallel orientation and a non-specific target sequence were tested as controls. Apparent binding constants for formation of the triplex were determined by quantitating electrophoretic band shifts. Binding of the single-stranded oligonucleotide probe sequence to the target led to an increase in the fluorescence anisotropy of acridine. The parallel orientation of the two identical sequence segments was confirmed by measurement of fluorescence resonance energy transfer between the acridine on the 5′-end of the probe strand as donor and BODIPY-Texas Red on the 3′-amino group of either strand of the target duplex as acceptor. There was full protection from OsO4-bipyridine modification of thymines in the probe strand of the triplex, in accordance with the presumed triplex formation, which excluded displacement of the homologous duplex strand by the probe–intercalator conjugate. The implications of these results for the existence of protein-independent parallel triplexes are discussed.

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.

Import of DNA into mammalian nuclei by proteins originating from a plant pathogenic bacterium

Import of DNA into mammalian nuclei is generally inefficient. Therefore, one of the current challenges in human gene therapy is the development of efficient DNA delivery systems. Here we tested whether bacterial proteins could be used to target DNA to mammalian cells. Agrobacterium tumefaciens, a plant pathogen, efficiently transfers DNA as a nucleoprotein complex to plant cells. Agrobacterium-mediated T-DNA transfer to plant cells is the only known example for interkingdom DNA transfer and is widely used for plant transformation. Agrobacterium virulence proteins VirD2 and VirE2 perform important functions in this process. We reconstituted complexes consisting of the bacterial virulence proteins VirD2, VirE2, and single-stranded DNA (ssDNA) in vitro. These complexes were tested for import into HeLa cell nuclei. Import of ssDNA required both VirD2 and VirE2 proteins. A VirD2 mutant lacking its C-terminal nuclear localization signal was deficient in import of the ssDNA–protein complexes into nuclei. Import of VirD2–ssDNA–VirE2 complexes was fast and efficient, and was shown to depended on importin α, Ran, and an energy source. We report here that the bacterium-derived and plant-adapted protein–DNA complex, made in vitro, can be efficiently imported into mammalian nuclei following the classical importin-dependent nuclear import pathway. This demonstrates the potential of our approach to enhance gene transfer to animal cells.

DNA packing in stable lipid complexes designed for gene transfer imitates DNA compaction in bacteriophage

The structure of complexes made from DNA and suitable lipids (lipoplex, Lx) was examined by cryo-electron microscopy (cryoEM). We observed a distinct concentric ring-like pattern with striated shells when using plasmid DNA. These spherical multilamellar particles have a mean diameter of 254 nm with repetitive spacing of 7.5 nm with striation of 5.3 nm width. Small angle x-ray scattering revealed repetitive ordering of 6.9 nm, suggesting a lamellar structure containing at least 12 layers. This concentric and lamellar structure with different packing regimes also was observed by cryoEM when using linear double-stranded DNA, single-stranded DNA, and oligodeoxynucleotides. DNA chains could be visualized in DNA/lipid complexes. Such specific supramolecular organization is the result of thermodynamic forces, which cause compaction to occur through concentric winding of DNA in a liquid crystalline phase. CryoEM examination of T4 phage DNA packed either in T4 capsides or in lipidic particles showed similar patterns. Small angle x-ray scattering suggested an hexagonal phase in Lx-T4 DNA. Our results indicate that both lamellar and hexagonal phases may coexist in the same Lx preparation or particle and that transition between both phases may depend on equilibrium influenced by type and length of the DNA used.

Bioactive and nuclease-resistant l-DNA ligand of vasopressin

In vitro selection experiments have produced nucleic acid ligands (aptamers) that bind tightly and specifically to a great variety of target biomolecules. The utility of aptamers is often limited by their vulnerability to nucleases present in biological materials. One way to circumvent this problem is to select an aptamer that binds the enantiomer of the target, then synthesize the enantiomer of the aptamer as a nuclease-insensitive ligand of the normal target. We have so identified a mirror-image single-stranded DNA that binds the peptide hormone vasopressin and have demonstrated its stability to nucleases and its bioactivity as a vasopressin antagonist in cell culture.

Developmental changes in DNA methylation of the two tobacco pollen nuclei during maturation

Changes in DNA methylation during tobacco pollen development have been studied by confocal fluorescence microscopy using a monoclonal anti-5-methylcytosine (anti-m5C) antibody and a polyclonal anti-histone H1 (anti-histone) antibody as an internal standard. The specificity of the anti-m5C antibody was demonstrated by a titration series against both single-stranded DNA and double-stranded DNA substrates in either the methylated or unmethylated forms. The antibody was found to show similar kinetics against both double- and single-stranded DNA, and the fluorescence was proportional to the amount of DNA used. No signal was observed with unmethylated substrates. The extent of methylation of the two pollen nuclei remained approximately constant after the mitotic division that gave rise to the vegetative and generative nuclei. However, during the subsequent development of the pollen, the staining of the generative nucleus decreased until it reached a normalized value of \documentclass[12pt]{minimal} \usepackage{wasysym} \usepackage{amsmath} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} \begin{equation*}\frac{1}{5}\end{equation*}\end{document} of that of the vegetative nucleus. The use of a confocal microscope makes these data independent of possible focusing artefacts. The anti-histone antibody was used as a control to show that, while the antibody staining directed against 5-methylcytosine changed dramatically during pollen maturation, the histone signal did not. We observed the existence of structural dimorphism amongst tobacco pollen grains, the majority having three pollen apertures and the rest with four. However, the methylation changes observed occurred to the same extent in both subclasses.