The single Cys2-His2 zinc finger domain of the GAGA protein flanked by basic residues is sufficient for high-affinity specific DNA binding.

Specific DNA binding to the core consensus site GAGAGAG has been shown with an 82-residue peptide (residues 310-391) taken from the Drosophila transcription factor GAGA. Using a series of deletion mutants, it was demonstrated that the minimal domain required for specific binding (residues 310-372) includes a single zinc finger of the Cys2-His2 family and a stretch of basic amino acids located on the N-terminal end of the zinc finger. In gel retardation assays, the specific binding seen with either the peptide or the whole protein is zinc dependent and corresponds to a dissociation constant of approximately 5 x 10(-9) M for the purified peptide. It has previously been thought that a single zinc finger of the Cys2-His2 family is incapable of specific, high-affinity binding to DNA. The combination of an N-terminal basic region with a single Cys2-His2 zinc finger in the GAGA protein can thus be viewed as a novel DNA binding domain. This raises the possibility that other proteins carrying only one Cys2-His2 finger are also capable of high-affinity specific binding to DNA.

p140/c-Abl that binds DNA is preferentially phosphorylated at tyrosine residues.

EP is a DNA element found in the enhancer and promoter regions of several cellular and viral genes. Previously, we have identified the DNA binding p140/c-Abl protein that specifically recognizes this element. Here we show that phosphorylation is essential for the p140/c-Abl DNA binding activity and for the formation of DNA-protein complexes. Furthermore, by 32P labeling of cells and protein purification, we demonstrate that in vivo the EP-DNA-associated p140/c-Abl is a tyrosine phosphoprotein. By employing two different c-Abl antibodies, we demonstrate the existence of two distinct c-Abl populations in cellular extracts. p140/c-Abl is quantitatively the minor population, is heavily phosphorylated at both serine and tyrosine residues, and is active in autophosphorylation reactions.

Agrobacterium VirE2 protein mediates nuclear uptake of single-stranded DNA in plant cells.

Agrobacterium genetically transforms plant cells by transferring a single-stranded DNA (ssDNA) copy of the transferred DNA (T-DNA) element, the T-strand, in a complex with Agrobacterium proteins VirD2, bound to the 5' end, and VirE2. VirE2 binds single-stranded nucleic acid cooperatively, fully coating the T-strand, and the protein localizes to the plant cell nucleus when transiently expressed. The coupling of ssDNA binding and nuclear localizing activities suggests that VirE2 alone could mediate nuclear localization of ssDNA. In this study, fluorescently labeled ssDNA accumulated in the plant cell nucleus specifically when microinjected as a complex with VirE2. Microinjected ssDNA alone remained cytoplasmic. Import of VirE2-ssDNA complex into the nucleus via a protein import pathway was supported by (i) the inhibition of VirE2-ssDNA complex import in the presence of wheat germ agglutinin or a nonhydrolyzable GTP analog, both known inhibitors of protein nuclear import, and (ii) the retardation of import when complexes were prepared from a VirE2 mutant impaired in ssDNA binding and nuclear import.

In vivo protein-DNA interactions at human DNA replication origin.

Protein-DNA interactions were studied in vivo at the region containing a human DNA replication origin, located at the 3' end of the lamin B2 gene and partially overlapping the promoter of another gene, located downstream. DNase I treatment of nuclei isolated from both exponentially growing and nonproliferating HL-60 cells showed that this region has an altered, highly accessible, chromatin structure. High-resolution analysis of protein-DNA interactions in a 600-bp area encompassing the origin was carried out by the in vivo footprinting technique based on the ligation-mediated polymerase chain reaction. In growing HL-60 cells, footprints at sequences homologous to binding sites for known transcription factors (members of the basic-helix-loop-helix family, nuclear respiratory factor 1, transcription factor Sp1, and upstream binding factor) were detected in the region corresponding to the promoter of the downstream gene. Upon conversion of cells to a nonproliferative state, a reduction in the intensity of these footprints was observed that paralleled the diminished transcriptional activity of the genomic area. In addition to these protections, in close correspondence to the replication initiation site, a prominent footprint was detected that extended over 70 nucleotides on one strand only. This footprint was absent from nonproliferating HL-60 cells, indicating that this specific protein-DNA interaction might be involved in the process of origin activation.

Specific cloning of human DNA as yeast artificial chromosomes by transformation-associated recombination.

DNA molecules undergoing transformation into yeast are highly recombinogenic, even when diverged. We reasoned that transformation-associated recombination (TAR) could be employed to clone large DNAs containing repeat sequences, thereby eliminating the need for in vitro enzymatic reactions such as restriction and ligation and reducing the amount of DNA handling. Gently isolated human DNA was transformed directly into yeast spheroplasts along with two genetically marked (M1 and M2) linearized vectors that contained a human Alu sequence at one end and a telomere sequence at the other end (Alu-CEN-M1-TEL and Alu-M2-TEL). Nearly all the M1-selected transformants had yeast artificial chromosomes (YACs) containing human DNA inserts that varied in size from 70 kb to > 600 kb. Approximately half of these had also acquired the unselected M2 marker. The mitotic segregational stability of YACs generated from one (M1) or two (M1 and M2) vector(s) was comparable, suggesting de novo generation of telomeric ends. Since no YACs were isolated when rodent DNAs or a vector lacking an Alu sequence was used, the YACs were most likely the consequence of TAR between the repeat elements on the vector(s) and the human DNA. Using the BLUR13 Alu-containing vector, we demonstrated that human DNA could be efficiently cloned from mouse cells that contained a single human chromosome 16. The distribution of cloned DNAs on chromosome 16 was determined by fluorescence in situ hybridization. We propose that TAR cloning can provide an efficient means for generating YACs from specific chromosomes and subchromosome fragments and that TAR cloning may be useful for isolating families of genes and specific genes from total genome DNA.

Catalytic editing properties of DNA polymerases.

Enzymatic incorporation of 2',3'-dideoxynucleotides into DNA results in chain termination. We report that 3'-esterified 2'-deoxynucleoside 5'-triphosphates (dNTPs) are false chain-terminator substrates since DNA polymerases, including human immunodeficiency virus reverse transcriptase, can incorporate them into DNA and, subsequently, use this new 3' end to insert the next correctly paired dNTP. Likewise, a DNA substrate with a primer chemically esterified at the 3' position can be extended efficiently upon incubation with dNTPs and T7 DNA polymerase lacking 3'-to-5' exonuclease activity. This enzyme is also able to use dTTP-bearing reporter groups in the 3' position conjugated through amide or thiourea bonds and cleave them to restore a DNA chain terminated by an amino group at the 3' end. Hence, a number of DNA polymerases exhibit wide catalytic versatility at the 3' end of the nascent DNA strand. As part of the polymerization mechanism, these capabilities extend the number of enzymatic activities associated with these enzymes and also the study of interactions between DNA polymerases and nucleotide analogues.

Antisense DNA downregulation of the ERBB2 oncogene measured by a flow cytometric assay.

A causal role has been inferred for ERBB2 overexpression in the etiology of breast cancer and other epithelial malignancies. The development of therapeutics that inhibit this tyrosine kinase cell surface receptor remains a high priority. This report describes the specific downregulation of ERBB2 protein and mRNA in the breast cancer cell line SK-BR-3 by using antisense DNA phosphorothioates. An approach was developed to examine antisense effects which allows simultaneous measurements of antisense dose and gene specific regulation on a per cell basis. A fluorescein isothiocyanate end-labeled tracer oligonucleotide was codelivered with antisense DNA followed by immunofluorescent staining for ERBB2 protein expression. Two-color flow cytometry measured the amount of both intracellular oligonucleotide and ERBB2 protein. In addition, populations of cells that received various doses of nucleic acids were physically separated and studied. In any given transfection, a 100-fold variation in oligonucleotide dosage was found. ERBB2 protein expression was reduced greater than 50%, but only in cells within a relatively narrow uptake range. Steady-state ERBB2 mRNA levels were selectively diminished, indicating a specific antisense effect. Cells receiving the optimal antisense dose were sorted and analyzed for cell cycle changes. After 2 days of ERBB2 suppression, breast cancer cells showed an accumulation in the G1 phase of the cell cycle.

Correlation of individual papilloma latency time with DNA adducts, 8-hydroxy-2'-deoxyguanosine, and the rate of DNA synthesis in the epidermis of mice treated with 7,12-dimethylbenz[alpha]anthracene.

The question was addressed whether the risk of cancer of an individual in a heterogeneous population can be predicted on the basis of measurable biochemical and biological variables postulated to be associated with the process of chemical carcinogenesis. Using the skin tumor model with outbred male NMRI mice, the latency time for the appearance of a papilloma was used as an indicator of the individual cancer risk. Starting at 8 weeks of age, a group of 29 mice was treated twice weekly with 20 nmol of 7,12-dimethylbenz[alpha]anthracene (DMBA) applied to back skin. The individual papilloma latency time ranged from 13.5 to 25 weeks of treatment. Two weeks after the appearance of the first papilloma in each mouse, an osmotic minipump delivering 5-bromo-2'-deoxyuridine was s.c. implanted and the mouse was killed 24 hr later. Levels of DMBA-DNA adducts, of 8-hydroxy-2'-deoxyguanosine, and various measures of the kinetics of cell division were determined in the epidermis of the treated skin area. The levels of 8-hydroxy-2'-deoxyguanosine and the fraction of cells in DNA replication (labeling index for the incorporation of 5-bromo-2'-deoxyuridine) were significantly higher in those mice that showed short latency times. On the other hand, the levels of DMBA-DNA adducts were lowest in animals with short latency times. The latter finding was rather unexpected but can be explained as a consequence of the inverse correlation seen for the labeling index: with each round of cell division, the adduct concentration is reduced to 50% because the new DNA strand is free of DMBA adducts until the next treatment. Under the conditions of this bioassay, therefore, oxygen radical-related genotoxicity and the rate of cell division, rather than levels of carcinogen-DNA adducts, were found to be of predictive value as indicators of an individual cancer risk.

Coiled bodies contain U7 small nuclear RNA and associate with specific DNA sequences in interphase human cells.

Coiled bodies (CBs) are nuclear organelles whose structures appear to be highly conserved in evolution. In rapidly cycling cells, they are typically located in the nucleoplasm but are often found in contact with the nucleolus. The CBs in human cells contain a unique protein, called p80-coilin. Studies on amphibian oocyte nuclei have revealed a protein within the "sphere" organelle that shares significant structural similarity to p80-coilin. Spheres and CBs are also highly enriched in small nuclear ribonucleoproteins and other RNA-processing components. We present evidence that, like spheres, CBs contain U7 small nuclear RNA (snRNA) and associate with specific chromosomal loci. Using biotinylated 2'-O-methyl oligonucleotides complementary to the 5' end of U7 snRNA and fluorescence in situ hybridization, we show that U7 is distributed throughout the nucleoplasm, excluding nucleoli, and is concentrated in CBs. Interestingly, we found that CBs often associate with subsets of the histone, U1, and U2 snRNA gene loci in interphase HeLa-ATCC and HEp-2 monolayer cells. However, in a strain of suspension-grown HeLa cells, called HeLa-JS1000, we found a much lower rate of association between CBs and snRNA genes. Possible roles for CBs in the metabolism of these various histone and snRNAs are discussed.

Fluorescence energy transfer dye-labeled primers for DNA sequencing and analysis.

Fluorescent dye-labeled DNA primers have been developed that exploit fluorescence energy transfer (ET) to optimize the absorption and emission properties of the label. These primers carry a fluorescein derivative at the 5' end as a common donor and other fluorescein and rhodamine derivatives attached to a modified thymidine residue within the primer sequence as acceptors. Adjustment of the donor-acceptor spacing through the placement of the modified thymidine in the primer sequence allowed generation of four primers, all having strong absorption at a common excitation wavelength (488 nm) and fluorescence emission maxima of 525, 555, 580, and 605 nm. The ET efficiency of these primers ranges from 65% to 97%, and they exhibit similar electrophoretic mobilities by gel electrophoresis. With argon-ion laser excitation, the fluorescence of the ET primers and of the DNA sequencing fragments generated with ET primers is 2- to 6-fold greater than that of the corresponding primers or fragments labeled with single dyes. The higher fluorescence intensity of the ET primers allows DNA sequencing with one-fourth of the DNA template typically required when using T7 DNA polymerase. With single-stranded M13mp18 DNA as the template, a typical sequencing reaction with ET primers on a commercial sequencer provided DNA sequences with 99.8% accuracy in the first 500 bases. ET primers should be generally useful in the development of other multiplex DNA sequencing and analysis methods.

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.

In vitro cleavage and joining at the viral origin of replication by the replication initiator protein of tomato yellow leaf curl virus.

Replication of the single-stranded DNA genome of geminiviruses occurs via a double-stranded intermediate that is subsequently used as a template for rolling-circle replication of the viral strand. Only one of the proteins encoded by the virus, here referred to as replication initiator protein (Rep protein), is indispensable for replication. We show that the Rep protein of tomato yellow leaf curl virus initiates viral-strand DNA synthesis by introducing a nick in the plus strand within the nonanucleotide 1TAATATT decreases 8AC, identical among all geminiviruses. After cleavage, the Rep protein remains bound to the 5' end of the cleaved strand. In addition, we show that the Rep protein has a joining activity, suggesting that it acts as a terminase, thus resolving the nascent viral single strand into genome-sized units.

NMR of enzymatically synthesized uniformly 13C15N-labeled DNA oligonucleotides.

A procedure for the enzymatic synthesis of uniformly 13C15N-labeled DNA oligonucleotides in milligram quantities for NMR studies is described. Deoxynucleotides obtained from microorganisms grown on 13C and 15N nutrient sources are enzymatically phosphorylated to dNTPs, and the dNTPs are incorporated into oligonucleotides using a 3'-5' exonuclease-deficient mutant of Klenow fragment of DNA polymerase I and an oligonucleotide template primer designed for efficient separation of labeled product DNA from unlabeled template. The labeling strategy has been used to uniformly label one or the other oligonucleotide strand in the DNA duplex dGGCAAAACGG.dCCGTTTTGCC in order to facilitate assignment and structure determination by NMR. Application of 15N and 13C heteronuclear NMR experiments to isotopically labeled DNA is presented.

DNA recombination is sufficient for retroviral transduction.

Oncogenic retroviruses carry coding sequences that are transduced from cellular protooncogenes. Natural transduction involves two nonhomologous recombinations and is thus extremely rare. Since transduction has never been reproduced experimentally, its mechanism has been studied in terms of two hypotheses: (i) the DNA model, which postulates two DNA recombinations, and (ii) the RNA model, which postulates a 5' DNA recombination and a 3' RNA recombination occurring during reverse transcription of viral and protooncogene RNA. Here we use two viral DNA constructs to test the prediction of the DNA model that the 3' DNA recombination is achieved by conventional integration of a retroviral DNA 3' of the chromosomal protooncogene coding region. For the DNA model to be viable, such recombinant viruses must be infectious without the purportedly essential polypurine tract (ppt) that precedes the 3' long terminal repeat (LTR) of all retroviruses. Our constructs consist of a ras coding region from Harvey sarcoma virus which is naturally linked at the 5' end to a retroviral LTR and artificially linked at the 3' end either directly (construct NdN) or by a cellular sequence (construct SU) to the 5' LTR of a retrovirus. Both constructs lack the ppt, and the LTR of NdN even lacks 30 nucleotides at the 5' end. Both constructs proved to be infectious, producing viruses at titers of 10(5) focus-forming units per ml. Sequence analysis proved that both viruses were colinear with input DNAs and that NdN virus lacked a ppt and the 5' 30 nucleotides of the LTR. The results indicate that DNA recombination is sufficient for retroviral transduction and that neither the ppt nor the complete LTR is essential for retrovirus replication. DNA recombination explains the following observations by others that cannot be reconciled with the RNA model: (i) experimental transduction is independent of the packaging efficiency of viral RNA, and (ii) experimental transduction may invert sequences with respect to others, as expected for DNA recombination during transfection.

Cell cycle-regulated nuclear import and export of Cdc47, a protein essential for initiation of DNA replication in budding yeast.

The CDC47 gene was isolated by complementation of a cdc47 temperature-sensitive mutant in Saccharomyces cerevisiae and was shown to encode a predicted polypeptide, Cdc47, of 845 aa. Cdc47 belongs to the Cdc46/Mcm family of proteins, previously shown to be essential for initiation of DNA replication. Using indirect immunofluorescence microscopy and subcellular fractionation techniques, we show that Cdc47 undergoes cell cycle-regulated changes in its subcellular localization. At mitosis, Cdc47 enters the nucleus, where it remains until soon after the initiation of DNA replication, when it is rapidly exported back into the cytoplasm. Cdc47 protein levels do not vary with the cell cycle, but expression of CDC47 and nascent synthesis of Cdc47 occur late in the cell cycle, coinciding with mitosis. Together, these results show that Cdc47 is not only imported into the nucleus at the end of mitosis but is also exported back into the cytoplasm at the beginning of S phase. The observation that Cdc47 is exported from the nucleus at the beginning of S phase has important implications for how initiation of DNA replication is controlled.