Binding of Hoechst 33258 and its Derivatives to DNA

Abstract

In the present work, we employed UV-VIS spectroscopy, fluorescence methods, and circular dichroism spectroscopy (CD) to study the interaction of dye Hoechst 33258, Hoechst 33342, and their derivatives to poly[d(AT)]·poly[d(AT)], poly(dA)·poly(dT), and DNA dodecamer with the sequence 5′-CGTATATATACG-3′. We identified three types of complexes formed by Hoechst 33258, Hoechst 33342, and methylproamine with DNA, corresponding to the binding of each drug in monomer, dimer, and tetramer forms. In a dimer complex, two dye molecules are sandwiched in the same place of the minor DNA groove. Our data show that Hoechst 33258, Hoechst 33342, and methylproamine also form complexes of the third type that reflects binding of dye associates (probably tetramers) to DNA. Substitution of a hydrogen atom in the ortho position of the phenyl ring by a methyl group has a little effect on binding of monomers to DNA. However it reduces strength of binding of tetramers to DNA. In contrast, a Hoechst derivative containing the ortho-isopropyl group in the phenyl ring exhibits a low affinity to poly(dA)·poly(dT) and poly[d(AT)]·poly[d(AT)] and binds to DNA only in the monomer form. This can be attributed to a sterical hindrance caused by the ortho-isopropyl group for side-by-side accommodation of two dye molecules in the minor groove. Our experiments show that mode of binding of Hoechst 33258 derivatives and their affinity for DNA depend on substituents in the ortho position of the phenyl ring of the dye molecule. A statistical mechanical treatment of binding of Hoechst 33258 and its derivatives to a polynucleotide lattice is described and used for determination of binding parameters of Hoechst 33258 and its derivatives to poly[d(AT)]·poly[d(AT)] and poly(dA)·poly(dT).     

Nrg1 functions as a global transcriptional repressor of glucose-repressed genes through its direct binding to the specific promoter regions

Nrg1 is a zinc finger protein involved in the glucose repression of several glucose-repressed genes such as STA1, SUC2, and GAL1. Although the molecular details of the Nrg1-mediated repression of STA1 have been partly characterized, it still remains largely unknown how Nrg1 regulates these multiple target genes. In this study, we show that Nrg1 mediates the glucose repression of SUC2 and HXT2 through its direct binding to the specific promoter regions; it binds to the −404 to −360 region of the SUC2 promoter and the −957 to −810 region of the HXT2 promoter. Nrg1 also interacts with the −380 to −250 region of the PCK1 promoter, suggesting that it might also contribute to the PCK1 repression. In addition, ChIP assays confirmed that Nrg1 associated with specific promoter regions of these glucose-repressed genes in vivo. Analysis of the DNA fragments to which it binds indicates that Nrg1 may recognize T/ACCCC sequence within the promoters of these glucose-repressed genes as well as in its own promoter. Collectively, our findings indicate that Nrg1 mediates the glucose repression of multiple genes through its direct binding to the specific promoter regions.     

The DrrAB Efflux System of Streptomyces peucetius Is a Multidrug Transporter of Broad Substrate Specificity

The soil bacterium Streptomyces peucetius produces two widely used anticancer antibiotics, doxorubicin and daunorubicin. Present within the biosynthesis gene cluster in S. peucetius is the drrAB operon, which codes for a dedicated ABC (ATP binding cassette)-type transporter for the export of these two closely related antibiotics. Because of its dedicated nature, the DrrAB system is believed to belong to the category of single-drug transporters. However, whether it also contains specificity for other known substrates of multidrug transporters has never been tested. In this study we demonstrate under both in vivo and in vitro conditions that the DrrAB system can transport not only doxorubicin but is also able to export two most commonly studied MDR substrates, Hoechst 33342 and ethidium bromide. Moreover, we demonstrate that many other substrates (including verapamil, vinblastine, and rifampicin) of the well studied multidrug transporters inhibit DrrAB-mediated Dox transport with high efficiency, indicating that they are also substrates of the DrrAB pump. Kinetic studies show that inhibition of doxorubicin transport by Hoechst 33342 and rifampicin occurs by a competitive mechanism, whereas verapamil inhibits transport by a non-competitive mechanism, thus suggesting the possibility of more than one drug binding site in the DrrAB system. This is the first in-depth study of a drug resistance system from a producer organism, and it shows that a dedicated efflux system like DrrAB contains specificity for multiple drugs. The significance of these findings in evolution of poly-specificity in drug resistance systems is discussed.     

Daunomycin-induced unfolding and aggregation of chromatin.

Using equilibrium dialysis and sedimentation velocity analysis, we have characterized the binding of the anti-tumor drug daunomycin to chicken erythrocyte chromatin before and after depletion of linker histones and to its constitutive DNA under several ionic strengths (5, 25, and 75 mM NaCl). The equilibrium dialysis experiments reveal that the drug binds cooperatively to both the chromatin fractions and to the DNA counterpart within the range of ionic strength used in this study. A significant decrease in the binding affinity was observed at 75 mM NaCl. At any given salt concentration, daunomycin exhibits higher binding affinity for DNA than for linker histone-depleted chromatin or chromatin (in decreasing order). Binding of daunomycin to DNA does not significantly affect the sedimentation coefficient of the molecule. This is in contrast to binding to chromatin and to its linker histone-depleted counterpart. In these instances, preferential binding of the drug to the linker DNA regions induces an unfolding of the chromatin fiber that is followed by aggregation, presumably because of histone-DNA interfiber interactions.     

Hoechst 33342 Is a Hidden “Janus” amongst Substrates for the Multidrug Efflux Pump LmrP

Multidrug transporters mediate the active extrusion of antibiotics and toxic ions from the cell. This reaction is thought to be based on a switch of the transporter between two conformational states, one in which the interior substrate binding cavity is available for substrate binding at the inside of the cell, and another in which the cavity is exposed to the outside of the cell to enable substrate release. Consistent with this model, cysteine cross-linking studies with the Major Facilitator Superfamily drug/proton antiporter LmrP from Lactococcus lactis demonstrated binding of transported benzalkonium to LmrP in its inward-facing state. The fluorescent dye Hoechst 33342 is a substrate for many multidrug transporters and is extruded by efflux pumps in microbial and mammalian cells. Surprisingly, and in contrast to other multidrug transporters, LmrP was found to actively accumulate, rather than extrude, Hoechst 33342 in lactococcal cells. Consistent with this observation, LmrP expression was associated with cellular sensitivity, rather than resistance to Hoechst 33342. Thus, we discovered a hidden “Janus” amongst LmrP substrates that is translocated in reverse direction across the membrane by binding to outward-facing LmrP followed by release from inward-facing LmrP. These findings are in agreement with distance measurements by electron paramagnetic resonance in which Hoechst 33342 binding was found to stabilize LmrP in its outward-facing conformation. Our data have important implications for the use of multidrug exporters in selective targeting of “Hoechst 33342-like” drugs to cells and tissues in which these transporters are expressed.     

The secondary multidrug transporter LmrP contains multiple drug interaction sites.

The secondary multidrug transporter LmrP of Lactococcus lactis mediates the efflux of Hoechst 33342 from the cytoplasmic leaflet of the membrane. Kinetic analysis of Hoechst 33342 transport in inside-out membrane vesicles of L. lactis showed that the LmrP-mediated H(+)/Hoechst 33342 antiport reaction obeyed Michaelis-Menten kinetics, with a low apparent affinity constant of 0.63 microM Hoechst 33342 (= 0.5 mmol Hoechst 33342/mol phospholipid). Several drugs significantly inhibited LmrP-mediated Hoechst 33342 transport through a direct interaction with the protein rather than through dissipation of the proton motive force or reduction of the membrane partitioning of Hoechst 33342. The characterization of the mechanism of inhibition of LmrP-mediated Hoechst 33342 transport indicated competitive inhibition by quinine and verapamil, noncompetitive inhibition by nicardipin and vinblastin, and uncompetitive inhibition by TPP(+). The three types of inhibition of LmrP-mediated Hoechst 33342 transport in inside-out membrane vesicles indicate for the first time the presence of multiple drug interaction sites in a secondary multidrug transporter.     

Binding of ArsR,the repressor of the Staphylococcus xylosus (pSX267) arsenic resistance operon to a sequence with dyad symmetry within the ars promoter

arsR, the first gene of the Staphylococcus xylosus (pSX267) arsenic/antimonite resistance (rs) operon encodes a negative regulatory protein, ArsR, which mediates inducibility of the resistances by arsenic and antimony compounds. ArsR, which has no obvious DNA-binding motif in its primary structure, was purified from an ArsR-overproducing Escherichia coli strain and identified as a DNA-binding protein by its behaviour in gel mobility shift assays. ArsR had a specific affinity for a 312 by DNA restriction fragment carrying the ars promoter; the minimum sequence complexed by ArsR was a 75 by polymerase chain reaction (PCR) fragment, which mainly comprised the ?35 and ?10 regions of the promoter. The effect of inducers on the DNA-binding activity of ArsR was examined by in vitro induction assays; only arsenite inhibited DNA-binding of the repressor. DNase I footprinting revealed two protected regions within the promoter region, spanning 23 and 9 nucleotides, respectively. Furthermore, a new cleavage site for DNase I between the protected regions was made accessible by binding of the repressor. The footprints cover a region of three inverted repeats located between the ?35 and ?10 motifs of the ars promoter. By high resolution footprinting with the hydroxy radical, five sites of close contact between the protein and DNA were identified.     

Transition state P-glycoprotein binds drugs and modulators with unchanged affinity,suggesting a concerted transport mechanism

The P-glycoprotein multidrug transporter is a plasma membrane efflux pump for hydrophobic natural products, drugs, and peptides, driven by ATP hydrolysis. Determination of the details of the catalytic cycle of P-glycoprotein is critical if we are to understand the mechanism of drug transport and design ways to inhibit it. It has been proposed that the vanadate-trapped transition state of P-glycoprotein (Pgp x ADP x V(i) x M(2+), where M(2+) is a divalent metal ion) has a very low affinity for drugs compared to resting state protein, thus leading to binding of substrate on the cytoplasmic side of the membrane and release of substrate to the extracellular medium (or the extracellular membrane leaflet). We have used several different fluorescence spectroscopic approaches to show that isolated purified P-glycoprotein, when trapped in a stable transition state with vanadate and either Co(2+)or Mg(2+), binds drugs with high affinity. For vinblastine, colchicine, rhodamine 123, and doxorubicin, the affinity of the vanadate-trapped transition state for drugs was only very slightly (less than 2-fold) lower than the binding affinity of resting state Pgp, whereas for the modulators cyclosporin A and verapamil and the substrate Hoechst 33342, the binding affinity was very similar for the two states. The drug binding affinity of the ADP-bound form of the transporter was also comparable to that of the unoccupied transporter. These results suggest that release of drug from the transporter during the catalytic cycle precedes formation of the transition state.     

Purification and characterization of MerR, the regulator of the broad-spectrum mercury resistance genes in Streptomyces lividans 1326

Streptomyces lividans 1326 carries inducible mercury resistance genes on the chromosome, which are arranged in two divergently transcribed operons. Expression of the genes is negatively regulated by the repressor MerR, which binds in the intercistronic region between the two operons. The merR gene was expressed in E. coli using a T7 RNA polymerase/promoter expression system, and MerR was purified to around 95% homogeneity by ammonium sulfate precipitation, gel filtration and affinity chromatography. Gel filtration showed that the native MerR is a dimer with a molecular mass of 31?kDa. Two DNA binding sites were identified in the intercistronic mer promoter region by footprinting experiments. No evidence for cooperativity in the binding of MerR to the adjacent operator sequences was observed in gel mobility shift assays. The dissociation constants (K_D) for binding of MerR were: binding site I, 8.5?×?10^?9?M; binding site II, 1.2?×?10^?8?M; and for the complete promoter/operator region 1?×?10^?8?M. The half-life of the MerR-DNA complex was 19.4?min and 18.8?min for binding site I and binding site II, respectively. The K_D value for binding of mercury(II)chloride to MerR, again determined by mobility shift assay, was 1.1?×?10^?7?M.     

Determination of affinity,stoichiometry and sequence selectivity of minor groove binder complexes with double-stranded oligodeoxynucleotides by electrospray ionization mass spectrometry

Electrospray mass spectrometry was evaluated regarding the reliability of the determination of the stoichiometries and equilibrium association constants from single spectra. Complexes between minor groove binders (Hoechst 33258, Hoechst 33342, DAPI, netropsin and berenil) and 12mer oligonucleotide duplexes with a central sequence (A/T)₄ flanked by G/C base pairs were chosen as model systems. To validate the electrospray ionization mass spectrometry (ESI-MS) method, comparisons were made with circular dichroism and fluorescence spectroscopy measurements. ESI-MS allowed the detection of minor (2 drug + DNA) species for Hoechst 33258, Hoechst 33342, DAPI and berenil with duplex d(GGGG(A/T)₄GGGG)· d(CCCC(A/T)₄CCCC), which were undetectable with the other techniques. Assuming that the duplexes and the complexes have the same electrospray response factors, the equilbrium association constants of the 1:1 and 2:1 complexes were determined by ESI-MS, and the values show a good quantitative agreement with fluorescence determined constants for Hoechst 33258 and Hoechst 33342. It is also shown that ESI-MS can quickly give reliable information on the A/T sequence selectivity of a drug: the signal of a complex is directly related to the affinity of the drug for that particular duplex. The potential of ESI-MS as a qualitative and quantitative affinity screening method is emphasized.