Isolation and characterization of two human transcription factor IIH (TFIIH)-related complexes: ERCC2/CAK and TFIIH.

Transcription factor IIH (TFIIH) is a multisubunit protein complex essential for both the initiation of RNA polymerase class II (pol II)-catalyzed transcription and nucleotide excision repair of DNA. Recent studies have shown that TFIIH copurifies with the cyclin-dependent kinase (cdk)-activating kinase complex (CAK) that includes cdk7, cyclin H, and p36/MAT1. Here we report the isolation of two TFIIH-related complexes: TFIIH* and ERCC2/CAK. TFIIH* consists of a subset of the TFIIH complex proteins including ERCC3 (XPB), p62, p44, p41, and p34 but is devoid of detectable levels of ERCC2 (XPD) and CAK. ERCC2/CAK was isolated as a complex that exhibits CAK activity that cosediments with the three CAK components (cdk7, cyclin H, and p36/MAT1) as well as the ERCC2 (XPD) protein. TFIIH* can support pol II-catalyzed transcription in vitro with lower efficiency compared with TFIIH. This TFIIH*-dependent transcription reaction was stimulated by ERCC2/CAK. The ERCC2/CAK and TFIIH* complexes are each active in DNA repair as shown by their ability to complement extracts prepared from ERCC2 (XPD)- and ERCC3 (XPB)-deficient cells, respectively, in supporting the excision of DNA containing a cholesterol lesion. These data suggest that TFIIH* and ERCC2/CAK interact to form the TFIIH holoenzyme capable of efficiently assembling the pol II transcription initiation complex and directly participating in excision repair reactions.

Human cyclin-dependent kinase-activating kinase exists in three distinct complexes.

Transcription factor IIH (TFIIH) is a multisubunit complex required for transcription and for DNA nucleotide excision repair. TFIIH possesses three enzymatic activities: (i) an ATP-dependent DNA helicase, (ii) a DNA-dependent ATPase, and (iii) a kinase with specificity for the carboxyl-terminal domain of RNA polymerase II. The kinase activity was recently identified as the cdk (cyclin-dependent kinase) activating kinase, CAK, composed of cdk7, cyclin H, and MAT-1. Here we report the isolation and characterization of three distinct CAK-containing complexes from HeLa nuclear extracts: CAK, a novel CAK-ERCC2 complex, and TFIIH. CAK-ERCC2 can efficiently associate with core-TFIIH to reconstitute holo-TFIIH transcription activity. We present evidence proposing a critical role for ERCC2 in mediating the association of CAK with core TFIIH subunits.

Multivalent DNA-binding properties of the HMG-1 proteins.

HMG-I proteins are DNA-binding proteins thought to affect the formation and function of transcription complexes. Each protein contains three DNA-binding motifs, known as AT-hooks, that bind in the minor groove of AT tracts in DNA. Multiple AT-hooks within a polypeptide chain should contact multiple AT tracts, but the rules governing these interactions have not been defined. In this study, we demonstrate that high-affinity binding uses two or three appropriately spaced AT tracts as a single multivalent binding site. These principles have implications for binding to regulatory elements such as the interferon beta enhancer, TATA boxes, and serum response elements.

Sequence-specific DNA-binding dominated by dehydration.

Fluorescence spectroscopy and isothermal titration calorimetry were used to study the thermodynamics of binding of the glucocorticoid receptor DNA-binding domain to four different, but similar, DNA-binding sites. The binding sites are two naturally occurring sites that differ in the composition of one base pair, i.e., an A-T to G-C mutation, and two sites containing chemical intermediates of these base pairs. The calorimetrically determined heat capacity change (Delta C(p)o(obs)) for glucocorticoid receptor DNA-binding domain binding agrees with that calculated for dehydration of solvent-accessible surface areas. A dominating effect of dehydration or solvent reorganization on the thermodynamics is also consistent with an observed linear relationship between observed enthalpy change (Delta Ho(obs)) and observed entropy change (Delta So(obs)) with a slope close to the experimental temperature. Comparisons with structural data allow us to rationalize individual differences between Delta Ho(obs) (and Delta So(obs)) for the four complexes. For instance, we find that the removal of a methyl group at the DNA-protein interface is enthalpically favorable but entropically unfavorable, which is consistent with a replacement by an ordered water molecule.

Sequence-specific arrest of primer extension on single-stranded DNA by an oligonucleotide-minor groove binder conjugate.

A minor groove binder (MGB) derivative (N-3-carbamoyl-1,2-dihydro-3H-pyrrolo[3,2-e]indole-7-carboxylate tripeptide; CDPI3) was covalently linked to the 5' or 3' end of several oligodeoxyribonucleotides (ODNs) totally complementary or possessing a single mismatch to M13mp19 single-stranded DNA. Absorption thermal denaturation and slot-blot hybridization studies showed that conjugation of CDPI3 to these ODNs increased both the specificity and the strength with which they hybridized. Primer extension of the same phage DNA by a modified form of phage T7 DNA polymerase (Sequenase) was physically blocked when a complementary 16-mer with a conjugated 5'-CDPI3 moiety was hybridized to a downstream site. Approximately 50% of the replicating complexes were arrested when the blocking ODN was equimolar to the phage DNA. Inhibition was unaffected by 3'-capping of the ODN with a hexanol group or by elimination of a preannealing step. Blockage was abolished when a single mismatch was introduced into the ODN or when the MGB was either removed or replaced by a 5'-acridine group. A 16-mer with a 3'-CDPI3 moiety failed to arrest primer extension, as did an unmodified 32-mer. We attribute the exceptional stability of hybrids formed by ODNs conjugated to a CDPI3 to the tethered tripeptide binding in the minor groove of the hybrid. When that group is linked to the 5' end of a hybridized ODN, it probably blocks DNA synthesis by inhibiting strand displacement. These ODNs conjugated to CDPI3 offer attractive features as diagnostic probes and antigene agents.

Protein synthesis editing by a DNA aptamer.

Potential errors in decoding genetic information are corrected by tRNA-dependent amino acid recognition processes manifested through editing reactions. One example is the rejection of difficult-to-discriminate misactivated amino acids by tRNA synthetases through hydrolytic reactions. Although several crystal structures of tRNA synthetases and synthetase-tRNA complexes exist, none of them have provided insight into the editing reactions. Other work suggested that editing required active amino acid acceptor hydroxyl groups at the 3' end of a tRNA effector. We describe here the isolation of a DNA aptamer that specifically induced hydrolysis of a misactivated amino acid bound to a tRNA synthetase. The aptamer had no effect on the stability of the correctly activated amino acid and was almost as efficient as the tRNA for inducing editing activity. The aptamer has no sequence similarity to that of the tRNA effector and cannot be folded into a tRNA-like structure. These and additional data show that active acceptor hydroxyl groups in a tRNA effector and a tRNA-like structure are not essential for editing. Thus, specific bases in a nucleic acid effector trigger the editing response.

Genes encoding the same three subunits of respiratory complex II are present in the mitochondrial DNA of two phylogenetically distant eukaryotes.

Although mitochondrial DNA is known to encode a limited number (<20) of the polypeptide components of respiratory complexes I, III, IV, and V, genes for components of complex II [succinate dehydrogenase (ubiquinone); succinate:ubiquinone oxidoreductase, EC 1.3.5.1] are conspicuously lacking in mitochondrial genomes so far characterized. Here we show that the same three subunits of complex II are encoded in the mitochondrial DNA of two phylogenetically distant eukaryotes, Porphyra purpurea (a photosynthetic red alga) and Reclinomonas americana (a heterotrophic zooflagellate). These complex II genes, sdh2, sdh3, and sdh4, are homologs, respectively, of Escherichia coli sdhB, sdhC, and sdhD. In E. coli, sdhB encodes the iron-sulfur subunit of succinate dehydrogenase (SDH), whereas sdhC and sdhD specify, respectively, apocytochrome b558 and a hydrophobic 13-kDa polypeptide, which together anchor SDH to the inner mitochondrial membrane. Amino acid sequence similarities indicate that sdh2, sdh3, and sdh4 were originally encoded in the protomitochondrial genome and have subsequently been transferred to the nuclear genome in most eukaryotes. The data presented here are consistent with the view that mitochondria constitute a monophyletic lineage.

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.

DNA-protein binding assays from a single sea urchin egg: a high-sensitivity capillary electrophoresis method.

A capillary electrophoresis method has been developed to study DNA-protein complexes by mobility-shift assay. This method is at least 100 times more sensitive than conventional gel mobility-shift procedures. Key features of the technique include the use of a neutral coated capillary, a small amount of linear polymer in the separation medium, and use of covalently dye-labeled DNA probes that can be detected with a commercially available laser-induced fluorescence monitor. The capillary method provides quantitative data in runs requiring < 20 min, from which dissociation constants are readily determined. As a test case we studied interactions of a developmentally important sea urchin embryo transcription factor, SpP3A2. As little as 2-10 x 10(6) molecules of specific SpP3A2-oligonucleotide complex were reproducibly detected, using recombinant SpP3A2, crude nuclear extract, egg lysates, and even a single sea urchin egg lysed within the capillary column.

Association with capsid proteins promotes nuclear targeting of simian virus 40 DNA.

All animal DNA viruses except pox virus utilize the cell nucleus as the site for virus reproduction. Yet, a critical viral infection process, nuclear targeting of the viral genome, is poorly understood. The role of capsid proteins in nuclear targeting of simian virus 40 (SV40) DNA, which is assessed by the nuclear accumulation of large tumor (T) antigen, the initial sign of the infectious process, was tested by two independent approaches: antibody interception experiments and reconstitution experiments. When antibody against viral capsid protein Vp1 or Vp3 was introduced into the cytoplasm, the nuclear accumulation of T antigen was not observed in cells either infected or cytoplasmically injected with virion. Nuclearly introduced anti-Vp3 IgG also showed the inhibitory effect. In the reconstitution experiments, SV40 DNA was allowed to interact with protein components of the virus, either empty particles or histones, and the resulting complexes were tested for the capability of protein components to target the DNA to the nucleus from cytoplasm as effectively as the targeting of DNA in the mature virion. In cells injected with empty particle-DNA, but not in minichromosome-injected cells, T antigen was observed as effectively as in SV40-injected cells. These results demonstrate that SV40 capsid proteins can facilitate transport of SV40 DNA into the nucleus and indicate that Vp3, one of the capsid proteins, accompanies SV40 DNA as it enters the nucleus during virus infection.

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 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.

Interaction of an alkylating camptothecin derivative with a DNA base at topoisomerase I-DNA cleavage sites.

DNA topoisomerase I (top1) is a ubiquitous nuclear enzyme. It is specifically inhibited by camptothecin, a natural product derived from the bark of the tree Camptotheca acuminata. Camptothecin and several of its derivatives are presently in clinical trial and exhibit remarkable anticancer activity. The present study is a further investigation of the molecular interactions between the drug and the enzyme-DNA complex. We utilized an alkylating camptothecin derivative, 7-chloromethyl-10,11-methylenedioxycamptothecin (7-ClMe-MDO-CPT), and compared its activity against calf thymus top1 in a DNA oligonucleotide containing a single top1 cleavage site with the activity of its nonalkylating analog, 7-ethyl-10,11-methylenedioxycamptothecin (7-Et-MDO-CPT). In the presence of top1, 7-ClMe-MDO-CPT produced a DNA fragment that migrated more slowly than the top1-cleaved DNA fragment observed with 7-Et-MDO-CPT. Top1 was unable to religate this fragment in the presence of high NaCl concentration or proteinase K at 50 degrees C. This fragment was resistant to piperidine treatment and was also formed with an oligonucleotide containing a 7-deazaguanine at the 5' terminus of the top1-cleaved DNA (base + 1). It was however cleaved by formic acid treatment followed by piperidine. These observations are consistent with alkylation of the +1 base (adenine or guanine) by 7-ClMe-MDO-CPT in the presence of top1 covalent complexes and provide direct evidence that camptothecins inhibit top1 by binding at the enzyme-DNA interface.

Transcription regulation by inflexibility of promoter DNA in a looped complex.

The gal operon of Escherichia coli is negatively regulated by repressor binding to bipartite operators separated by 11 helical turns of DNA. Synergistic binding of repressor to separate sites on DNA results in looping, with the intervening DNA as a topologically closed domain containing the two promoters. A closed DNA loop of 11 helical turns, which is in-flexible to torsional changes, disables the promoters either by resisting DNA unwinding needed for open complex formation or by impeding the processive DNA contacts by an RNA polymerase in flux during transcription initiation. Interaction between two proteins bound to different sites on DNA modulating the activity of the intervening segment toward other proteins by allostery may be a common mechanism of regulation in DNA-multiprotein complexes.