Proposed mechanism for the stabilization of nuclear receptor DNA binding via protein dimerization.

Hepatocyte nuclear factor 4 (HNF-4) defines a new subgroup of nuclear receptors that exist in solution and bind DNA exclusively as homodimers. We recently showed that the putative ligand binding domain (LBD) of HNF-4 is responsible for dimerization in solution and prevents heterodimerization with other receptors. In this report, the role of the LBD in DNA binding by HNF-4 is further investigated by using electrophoretic mobility shift analysis. A comparison of constructs containing either the DNA binding domain (DBD) alone or the DBD plus the LBD of HNF-4 showed that dimerization via the DBD was sufficient to provide nearly the full DNA binding affinity of the full-length HNF-4. In contrast, dimerization via the DBD was not sufficient to produce a stable protein-DNA complex, whereas dimerization via the LBD increased the half-life of the complex by at least 100-fold. Circular permutation analysis showed that full-length HNF-4 bent DNA by approximately 80 degrees while the DBD bent DNA by only 24 degrees. Nonetheless, analysis of other constructs indicated that the increase in stability afforded by the LBD could be explained only partially by an increased ability to bend DNA. Coimmunoprecipitation studies, on the other hand, showed that dimerization via the LBD produced a protein-protein complex that was much more stable than the corresponding protein-DNA complex. These results led us to propose a model in which dimerization via the LBD stabilizes the receptor on DNA by converting an energetically favorable two-step dissociation event into an energetically unfavorable single-step event. Implications of this one-step model for other nuclear receptors are discussed.     

General mechanism for RecA protein binding to duplex DNA

RecA protein binding to duplex DNA occurs by a multi-step process. The tau analysis, originally developed to examine the binding of RNA polymerase to promoter DNA, is adapted here to study two kinetically distinguishable reaction segments of RecA-double stranded (ds) DNA complex formation in greater detail. One, which is probably a rapid preequilibrium in which RecA protein binds weakly to native dsDNA, is found to have the following properties: (1) a sensitivity to pH, involving a net release of approximately one proton; (2) a sensitivity to salts; (3) little or no dependence on temperature; (4) little or no dependence on DNA length. The second reaction segment, the rate-limiting nucleation of nucleoprotein filament formation accompanied by partial DNA unwinding, is found to have the following properties: (1) a sensitivity to pH, involving a net uptake of approximately three protons; (2) a sensitivity to salts; (3) a relatively large dependence on temperature, with an Arrhenius activation energy of 39 kcal mol(-1); (4) a sensitivity to DNA topology; (5) a dependence on DNA length. These results contribute to a general mechanism for RecA protein binding to duplex DNA, which can provide a rationale for the apparent preferential binding to altered DNA structures such as pyrimidine dimers and Z-DNA.     

RFP is a DNA binding protein associated with the nuclear matrix.

We reported that the RFP gene encodes a protein with putative zinc finger domains and was involved in the activation of the ret proto-oncogene. To further characterize the RFP protein, we developed a polyclonal antibody against the product synthesized from a fragment of the RFP cDNA expressed in Escherichia coli. Western blot analysis showed that RFP was identified as a 58 kDa protein in cell lysates from four human and rodent cell lines and from mouse testis. In addition, a unique 68 kDa protein was detected in the testis. Using AH7974 (rat ascites hepatoma) and Raji (human Burkitt lymphoma) cells, we demonstrated strong association of RFP with the nuclear matrix. Furthermore, RFP solubilized from the nuclear matrix had DNA-binding activity although it appears to bind more preferentially to double-stranded DNA than to single-stranded DNA. These results thus suggest that RFP may play a role in molecular processes which occur in the nuclear matrix.     

Analysis of bisdioxopiperazine dexrazoxane binding to human DNA topoisomerase II alpha: decreased binding as a mechanism of drug resistance

Topoisomerase II is an ATP-operated clamp that effects topological changes by capturing a double stranded DNA segment and transporting it through another DNA molecule. Despite the extensive use of topoisomerase II-targeted drugs in cancer chemotherapy and the impact of drug resistance on the efficacy of treatment, much remains unknown concerning the interactions between these agents and topoisomerase II. To identify the interaction of the bisdioxopiperazine dexrazoxane (ICRF-187) with topoisomerase II, we developed a rapid gel-filtration assay and characterized the binding of ((3)H)-dexrazoxane to human topoisomerase II alpha. Dexrazoxane binds to human topoisomerase II alpha in the presence of DNA and ATP with an apparent K(d) of 23 microM and a stoichiometry of 1 drug molecule per enzyme dimer. Various N-terminal single amino acid substitutions in human topoisomerase II alpha that were previously shown to confer specific bisdioxopiperazine resistance either totally abolished drug binding or resulted in less efficient binding. The effect of the various mutations on drug binding correlated well with their effect on drug resistance in vivo and in vitro. Interestingly, an altered active site tyrosine mutant of human topoisomerase II alpha, which is incapable of carrying out DNA strand passage, was unable to bind dexrazoxane, which agrees with the drug's proposed mechanism of action late in the topoisomerase II catalytic cycle. The direct correlation between the level of drug binding and dexrazoxane resistance is consistent with a decreased drug binding mechanism of action for these dexrazoxane resistance conferring mutations.     

Increased metallothionein gene expression in 5-aza-2'-deoxycytidine-induced resistance to cadmium cytotoxicity

The pyrimidine analog, 5-azacytidine (AZA-CR), has been shown to increase the expression of the metallothionein (MT) gene and to induce tolerance to cadmium toxicity. Since incorporation into DNA of AZA-CR appears to be required for this effect, the deoxynucleoside of AZA-CR should also be effective. Therefore, this study was undertaken to assess the effect of 5-aza-2'-deoxycytidine (AZA-CdR) pretreatment on cadmium-induced cytotoxicity and MT expression in cultured cells. TRL 1215 cells in log phase of growth were exposed to AZA-CdR (0.4, 0.8, 4.0, 8.0 microM) followed 48 h later by the addition of cadmium (10 microM). MT concentrations were measured 24 h after the addition of cadmium. AZA-CdR alone caused modest, dose-related increases in MT levels (2.3-fold maximum), while cadmium alone resulted in a 9.5-fold increase. Pretreatment with AZA-CdR in combination with cadmium caused a 19--24-fold increase in cellular MT at all doses of AZA-CdR. Addition of the DNA synthesis inhibitor, hydroxyurea (HU), to the incubation medium during AZA-CdR exposure prevented the enhancing effect of the analog on cadmium induction of MT accumulation. Time course studies revealed that AZA-CdR pretreatment reduced the time required for cadmium to induce MT levels from 4--8 h to 0--2 h. AZA-CdR pretreated cells placed in suspension with cadmium (125 microM) showed a marked reduction in cadmium-induced cytotoxicity as reflected by reduced glutamic-oxaloacetic transaminase (GOT) loss. Uptake studies showed that AZA-CdR pretreatment had no effect on cadmium transport during the initial phases of exposure, indicating that an alteration in the toxicokinetics of the metal did not account for the reduction in toxicity. AZA-CdR did, however, cause hypomethylation of the MT-I gene. These results suggest that AZA-CdR pretreatment induces tolerance to cadmium toxicity by increasing the genetic expression of MT possibly through hypomethylation of the MT gene.     

5-Methylcytosine containing CG decamer as Z-DNA embedded sequence for a potential Z-DNA binding protein probe

Abstract

Attempting to elucidate biological significance of the left-handed Z-DNA is a research challenge due to Z-DNA potential role in many diseases. Discovery of Z-DNA binding proteins has ignited the interest in search for Z-DNA functions. Biosensor with Z-DNA forming probe can be useful to study the interaction between Z-DNA conformation and Z-DNA binding proteins. In this study, 5-methylcytosine (^mC) containing CG decamers were characterized for their suitability to form Z-DNA and to be used in Z-DNA forming probe. The 5′-thiol oligonucleotide embedded with 5′-^mCG^mCG^mCG^mCG^m CG-3′ was designed and developed as a potential Z-DNA forming probe for Z-DNA binding protein screening.     

Defective DNA repair as a potential mechanism for the rapid development of drug resistance in Plasmodium falciparum

The development and spread of highly drug-resistant parasites pose a central problem in the control of malaria.Understanding mechanisms that regulate genomic stability, such as DNA repair, in drug-resistant parasites and during drug treatment may help determine whether this rapid onset of resistance is due to an increase in the rate at which resistance-causing mutations are generated. This is the first report to demonstrate DNA repair activities from the malaria-causing parasite Plasmodium falciparum that are specific for ultraviolet light-induced DNA damage. The efficiency of DNA repair differs dramatically among P. falciparum strains with varying drug sensitivities. Most notable is the markedly reduced level of repair in the highly drug-resistant W2 isolate, which has been shown to develop resistance to novel drugs at an increased rate when compared to drug-sensitive strains. Additionally, the antimalarial drug chloroquine and other quinoline-like compounds interfered with the DNA synthesis step of the repair process, most likely a result of direct binding to repair substrates. We propose that altered DNA repair, either through defective repair mechanisms or drug-mediated inhibition, may contribute to the accelerated development of drug resistance in the parasite.     

Single-stranded bacteriophage T5 stO DNA as template for in vitro fMet-tRNA binding to ribosomes and protein synthesis.

Good evidence is provided that fMet-tRNA binding and aminoacid incorporation, when single-stranded DNA is used instead of mRNA in an E. coli cell-free system, are strictly dependent on the magnesium concentration. Ten sites homologous to the initiation sites of translation can be detected on denatured T5 stO DNA when using ribosomes and initiation factors from uninfected E. coli F. In S-30 extracts, at high magnesium concentrations and in the presence of neomycin, initiation of the translation of denatured T5 stO DNA begins anywhere on the molecule, and yet high molecular weight polypeptides are synthesized. The template potentiality of the denatured T5 stO DNA decreased when using ribosomes plus initiation factors and crude extracts from T5 stO-infected bacteria. By in vitro formation of initiation complexes sites analogous to initiation sites of translation were localized on T5 stO DNA molecules using single-stranded fragments separated by sedimentation in alkaline sucrose gradient.     

Multi-site binding of human nuclear protein to the Alu-family repeated DNA

Nuclear protein which selectively binds to the Alu-family DNA repeat (AFR, Blur8) is partially purified from human HeLa cells using a gel retention assay. At low protein concentrations only a single complex of the protein with AFR is formed (CII). Increasing protein concentrations lead to the gradual disappearance of CII, being replaced by complexes with higher (CI) and lower (CIII, CIV) electrophoretic mobilities. Differential binding of AFR restriction subfragments indicates that multiple protein-binding sites are present within AFR. We discuss two models explaining the anomalous electrophoretic mobility of CII by DNA bending or looping upon cooperative multi-site binding of the protein to AFR.     

Cloning of a complementary DNA encoding an 80 kilodalton nuclear cap binding protein.

It has been shown that the monomethylated cap structure plays important roles in nuclear events. The cap structure has been implicated in the enhancement of pre-mRNA splicing. More recently, this structure has also been suggested to facilitate RNA transport from the nucleus to the cytoplasm. We have previously identified and purified an 80kD Nuclear Cap Binding Protein (NCBP) from a HeLa cell nuclear extract, which could possibly mediate these nuclear activities. In this report, we describe cloning of complementary DNA (cDNA) encoding NCBP. The partial protein sequences of NCBP were determined, and the full-length cDNA of NCBP was isolated from HeLa cDNA libraries. This cDNA encoded an open reading frame of 790 amino acids with a calculated molecular mass of 91,734 daltons, which contained most of the determined protein sequences. However, the protein sequence had no significant homology to any known proteins. Transfection experiments demonstrated that the epitope-tagged NCBP, transiently expressed in HeLa cells, was localized exclusively in the nucleoplasm. Similar experiments using a truncated NCBP cDNA indicated that this nuclear localization activity is conferred by the N-terminal 70 amino-acid region.     

Differential binding of Escherichia coli McrA protein to DNA sequences that contain the dinucleotide m5CpG

The Escherichia coli McrA protein, a putative C⁵-methylcytosine/C⁵-hydroxyl methylcytosine-specific nuclease, binds DNA with symmetrically methylated HpaII sequences (Cm5CGG), but its precise recognition sequence remains undefined. To determine McrA’s binding specificity, we cloned and expressed recombinant McrA with a C-terminal StrepII tag (rMcrA-S) to facilitate protein purification and affinity capture of human DNA fragments with m5C residues. Sequence analysis of a subset of these fragments and electrophoretic mobility shift assays with model methylated and unmethylated oligonucleotides suggest that N(Y > R) m5CGR is the canonical binding site for rMcrA-S. In addition to binding HpaII-methylated double-stranded DNA, rMcrA-S binds DNA containing a single, hemimethylated HpaII site; however, it does not bind if A, C, T or U is placed across from the m5C residue, but does if I is opposite the m5C. These results provide the first systematic analysis of McrA’s in vitro binding specificity.     

Adenovirus type 5 DNA binding protein stimulates binding of DNA polymerase to the replication origin

The adenovirus (Ad) DNA-binding protein (DBP) is essential for the elongation phase of Ad DNA replication by unwinding the template in an ATP-independent fashion, employing its capacity to form multimers. DBP also enhances the rate of initiation, with the highest levels obtained at low concentrations of Ad DNA polymerase (Pol). Here, we show that stimulation of initiation depends on the template conformation. Maximal stimulation, up to 15-fold, is observed on double-stranded or viral TP-containing origins. The stimulation is reduced on partially single-stranded origins and DBP does not enhance initiation any more once the origin is completely unwound. This suggests a role for DBP in origin unwinding that is comparable to its unwinding capacity during elongation. However, mutant DBP proteins defective in unwinding and elongation can still enhance initiation on ds templates. DBP also stimulates the binding of nuclear factor I (NFI) to the origin and lowers the K(m) for coupling of the first nucleotide to the precursor terminal protein by Pol. Mobility shift experiments reveal that DBP stimulates the binding of Pol on double-stranded origin and nonorigin DNA but not on single-stranded DNA. This effect is specific for DBP and is also seen with other DNA Pols. Our results suggest that, rather than by origin unwinding, DBP enhances initiation by modulating the origin conformation such that DNA Pol can bind more efficiently.     

Characterization of a mitochondrial protein binding to single-stranded DNA.

A DNA-binding protein from Xenopus laevis oocyte mitochondria which has been found associated with the D-loop also shows a strong preference for single-stranded DNA. The binding to polynucleotides is dependent on the base composition, but no sequence specificity was found. This protein, called mtSSB, binds tightly and cooperatively to single-stranded DNA. By its amino-acid composition and its binding properties it appears to be similar to the single-stranded DNA-binding proteins found in prokaryotes.     

Differential binding of methyl benzimidazol-2-yl carbamate to fungal tubulin as a mechanism of resistance to this antimitotic agent in mutant strains of Aspergillus nidulans

The antimitotic compound methyl benzimidazol-2-yl carbamate (MBC) formed a complex in vitro with a protein present in mycelial extracts of fungi. The binding protein of Aspergillus nidulans showed a set of properties which is unique for tubulin. Binding occurred rapidly at 4 degrees C and was competitively inhibited by oncodazole and colchicine. Other inhibitors of microtubule function such as podophyllotoxin, vinblastine sulfate, melatonin, and griseofulvin did not interfere with binding of MBC. Electrophoretic analysis of partially purified preparations of the binding protein revealed the presence of proteins with similar mobilities as mammalian tubulin monomers. Hence it is concluded that the binding protein is identical with fungal tubulin. The effect of MBC on mycelial growth of mutant strains of A. nidulans was positively correlated with the affinity of the binding sites for this compound. The apparent binding constant for MBC and tubulin from a wild type was estimated at 4.5 X 10(5), from a resistant strain at 3.7 X 10(4), and from a strain with increased sensitivity to MBC at 1.6 X 10(6) liters/mol. Mutants showing resistance and increased sensitivity to MBC are candidates to have alterations in tubulin structure. Affinity of tubulin for MBC is probably a common mechanism of resistance to this compound in fungi. Low affinity of tubulin for MBC is probably a common mechanism of resistance binding constant of 2.5 X 10(3) liters/mol.