The two modes of binding of Ru(phen)(2)dppz(2+) to DNA: thermodynamic evidence and kinetic studies

The binding of Ru(phen)(2)dppz(2+) (dppz=dipyrido[3,2-a:2',3'-c]phenazine) to DNA was investigated at pH 7.0 and 25 degrees C using stopped-flow and spectrophotometric methods. Equilibrium measurements show that two modes of binding, whose characteristics depend on the polymer to dye ratio (C(P)/C(D)), are operative. The binding mode occurring for values of C(P)/C(D) higher than 3 exhibits positive cooperativity, which is confirmed by kinetic experiments. The reaction parameters are K=2 x 10(3)M(-1), omega=550, n=1, k(r)=(1.9+/-0.5) x 10(7)M(-1)s(-1) and k(d)=(9.5+/-2.5)x10(3)s(-1) at I=0.012 M. The results are discussed in terms of prevailing surface interaction with DNA grooves accompanied by partial intercalation of the dppz residue. The other binding mode becomes operative for C(P)/C(D)<3 and the equilibria analysis shows this is an ordinary intercalation mode (K=1.3 x 10(6) M(-1), n=1.5 at I=0.012 M and K=2 x 10(5) M(-1), n=1.2 at I=0.21 M). Similar behaviour is displayed by double-stranded poly(A).     

Minor groove binding DNA ligands with expanded A/T sequence length recognition, selective binding to bent DNA regions and enhanced fluorescent properties

DNA minor groove ligands provide a paradigm for double-stranded DNA recognition, where common structural motifs provide a crescent shape that matches the helix turn. Since minor groove ligands are useful in medicine, new ligands with improved binding properties based on the structural information about DNA-ligand complexes could be useful in developing new drugs. Here, two new synthetic analogues of AT specific Hoechst 33258 5-(4-methylpiperazin-1-yl)-2-[2'-(3,4-dimethoxyphenyl)-5'-benzimidazolyl] benzimidazole (DMA) and 5-(4-methylpiperazin-1-yl)-2-[2'[2'-(4-hydroxy-3-methoxyphenyl)-5' '-benzimidazolyl]-5'-benzimidazolyl] benzimidazole (TBZ) were evaluated for their DNA binding properties. Both analogues are bisubstituted on the phenyl ring. DMA contains two ortho positioned methoxy groups, and TBZ contains a phenolic group at C-4 and a methoxy group at C-3. Fluorescence yield upon DNA binding increased 100-fold for TBZ and 16-fold for DMA. Like the parent compound, the new ligands showed low affinity to GC-rich (K approximately 4 x 10(7) M(-1)) relative to AT-rich sequences (K approximately 5 x 10(8) M(-1)), and fluorescence lifetime and anisotropy studies suggest two distinct DNA-ligand complexes. Binding studies indicate expanded sequence recognition for TBZ (8-10 AT base pairs) and tighter binding (DeltaT(m) of 23 degrees C for d (GA(5)T(5)C). Finally, EMSA and equilibrium binding titration studies indicate that TBZ preferentially binds highly hydrated duplex domains with altered A-tract conformations d (GA(4)T(4)C)(2) (K= 3.55 x 10(9) M(-1)) and alters its structure over d (GT(4)A(4)C)(2) (K = 3.3 x 10(8) M(-1)) sequences. Altered DNA structure and higher fluorescence output for the bound fluorophore are consistent with adaptive binding and a constrained final complex. Therefore, the new ligands provide increased sequence and structure selective recognition and enhanced fluorescence upon minor groove binding, features that can be useful for further development as probes for chromatin structure stability.     

Tuning of intercalation and electron-transfer processes between DNA and acridinium derivatives through steric effects

A series of acridinium derivatives 1-6, wherein steric factors have been varied systematically through substitution at the 9 position of the acridine ring, have been synthesized and their DNA interactions have been investigated by various biophysical techniques. The unsubstituted and methylacridinium derivatives 1 and 2 and the o-tolylacridinium derivative 6 exhibited high fluorescence quantum yields (Phi(f)() congruent with 1) and lifetimes (tau = 35, 34, and 25 ns, respectively), when compared with the arylacridinium derivatives 3-5. The acridinium derivatives 1 and 2 showed high DNA binding affinity (K = 7.3-7.7 x 10(5) M(-)(1)), when compared to the arylacridinium derivatives 3-5 (K = 6.9-10 x 10(4) M(-)(1)). DNA melting and viscosity studies establish that in the case of the aryl-substituted systems, the efficiency of DNA binding is in the order, phenyl > p-tolyl > m-tolyl > o-tolyl derivative. The increase in steric crowding around the acridine ring hinders the DNA binding interactions and thereby leads to negligible binding as observed in the case of 6 (o-tolyl derivative). These results indicate that a subtle variation in the substitution pattern has a profound influence on the photophysical and DNA interactions. Further, they demonstrate that pi-stacking interactions of the ligands with DNA are essential for efficient electron transfer between the DNA bases and the ligands. These water soluble and highly fluorescent molecules which differ in their DNA binding mode can act as models to study various DNA-ligand interactions.     

Interactions of DNA binding ligands with PNA-DNA hybrids.

The interactions of two representative mixed-sequence (one with an AT-stretch) PNA-DNA duplexes (10 or 15 base-pairs) and a PNA2/DNA triplex with the DNA binding reagents distamycin A, 4',6-diamidino-2-phenylindole (DAPI), ethidium bromide, 8-methoxy-psoralen and the delta and lambda enantiomers of Ru(phen)2-dppz2+ have been investigated using optical spectroscopic methods. The behaviour of these reagents versus two PNA-PNA duplexes has also been investigated. With triple helical poly(dA)/(H-T10-Lys-NH2)2 no significant intercalative binding was detected for any of the DNA intercalators, whereas DAPI, a DNA minor groove binder, was found to exhibit a circular dichroism with a positive sign and amplitude consistent with minor groove binding. Similarly, a PNA-DNA duplex containing a central AATA motif, a typical minor groove binding site for the DNA minor groove binders distamycin A and DAPI, showed binding for both of these drugs, though with strongly reduced affinity. No important interactions were found for any of the ligands with a PNA-DNA duplex consisting of a ten base-pair mixed purine-pyrimidine sequence with only two AT base-pairs in the centre. Nor did any of the ligands show any detectable binding to the PNA-PNA duplexes (one containing an AATT motif). Various PNA derivatives with extentions of the backbone, believed to increase the flexibility of the duplex to opening of an intercalation slot, were tested for intercalation of ethidium bromide or 8-methoxypsoralen into the mixed sequence PNA-DNA duplex, however, without any observation of improved binding. The importance of the ionic contribution of the deoxyribose phosphate backbone, versus interactions with the nucleobases, for drug binding to DNA is discussed in the light of these findings.     

DNA binding and cleavage properties of certain tetrammine ruthenium(II) complexes of modified 1,10-phenanthrolines--effect of hydrogen-bonding on DNA-binding affinity

A series of ruthenium(II) mixed ligand complexes of the type [Ru(NH(3))(4)(L)](2+), where L=imidazo[4,5-f][1,10]phenanthroline (ip), 2-phenylimidazo[4,5-f][1,10]phenanthroline (pip), 2-(2-hydroxyphenyl)imidazo[4,5-f][1,10]phenanthroline (hpip), 4,7-diphenyl-1,10-phenanthroline (dip), naphtha[2,3-a]dipyrido[3,2-h:2',3'-f]phenazine-5,18-dione (qdppz), 5,18-dihydroxynaphtho[2,3-a]dipyrido[3,2-H:2',3'-f]phenazine (hqdppz), have been isolated and characterized. The interaction of these complexes with calf thymus DNA (CT DNA) has been explored by using absorption, emission, and circular dichroic spectral methods, thermal denaturation studies and viscometry. All these studies suggest the involvement of the modified phenanthroline 'face' rather than the ammonia 'face' of the complexes in DNA binding. An intercalative mode of DNA binding, which involves the insertion of the modified phenanthroline ligands in between the base pairs, is suggested. The results from absorption spectral titration and circular dichroism (CD), thermal denaturation and viscosity experiments indicate that the qdppz and hqdppz complexes (K(b) approximately 10(6) and Delta T(m)=11-13 degrees C) bind more avidly than the ip, pip and hpip complexes (K(b) approximately 10(5), Delta T(m)=6-8 degrees C). Intramolecular hydrogen bonding in the hpip and hqdppz complexes increases the surface area of the intercalating diimines and enhances the DNA binding affinity substantially. The ammonia co-ligands of the complexes are possibly involved in hydrogen bonding with the intrastrand nucleobases to favour intercalation of the extended aromatic ligands. Circular dichroism spectral studies reveal that all the complexes effect certain structural changes on DNA duplex; [Ru(NH(3))(4)(ip)](2+) induces a B to A transition while [Ru(NH(3))(4)(qdppz)](2+) a B to Psi conformational change on CT DNA. Cleavage efficiency of the complexes were determined using pBR322 supercoiled plasmid DNA. All the complexes, except hqdppz complex, promote the cleavage of supercoiled plasmid (form I) to relaxed circular form (form II).     

Syntheses and DNA-binding studies of two ruthenium(II) complexes containing one ancillary ligand of bpy or phen: [Ru(bpy)(pp[2,3]p)2](ClO4)2 and [Ru(phen)(pp[2,3]p)2](ClO4)2

Two new ruthenium(II) complexes of [Ru(bpy)(pp[2,3]p)2](ClO4)2 and [Ru(phen)(pp[2,3]p)2](ClO4)(2) (bpy=2,2'-bipyridine, phen=1,10-phenanthroline, pp[2,3]p=pyrido[2',3':5,6]pyrazino[2,3-f][1,10]phenanthroline) have been synthesized and characterized by elemental analysis and 1H NMR spectra. The calf thymus DNA-binding properties of the two complexes were investigated by UV-visible and emission spectroscopy, competitive binding experiments with ethidium bromide and viscosity measurements. The results indicate that the two complexes intercalate between the base pairs of the DNA tightly with intrinsic DNA-binding constants of 3.08 x 10(6) and 6.53 x 10(6) M(-1) in buffered 50 mM NaCl, respectively, which are much larger than 6.9 x 10(5) M(-1) for [Ru(bpy)2(pp[2,3]p)](ClO4)2 containing two ancillary ligands of bpy.     

Terpene conjugates of diaminedichloridoplatinum(II) complexes: antiproliferative effects in HL-60 leukemia, 518A2 melanoma, and HT-29 colon cancer cells

Twenty-eight [6-(aminomethyl)nicotinate]dichloridoplatinum(II) complexes 1 esterified with various terpene alcohols either directly or via alkyl spacers were tested for antiproliferative activity in human 518A2 melanoma and HL-60 leukemia cells. Generally, conjugates with menthanes and polycyclic sesquiterpenes attached via propane-1,2-diyl spacers were most active. In the melanoma cells, the propane-1,2-diyl-spacered conjugates of (-)-menthol (1a(2')), (+)-neomenthol (1b(2')), (-)-carvomenthol (1h(2')), and (-)-isolongifolol (1n(2')) displayed growth inhibition at IC(50)<4 microM which is ten times smaller than that of cisplatin. The stationary diamino ligand was also crucial. The (-)-menthyl ester complexes with 2,3-diaminopropanoate (9a) and 2,4-diaminobutanoate (10a) ligands caused a greater and persistent growth inhibition in HT-29 colon cancer cells upon long-term exposure when compared to the 6-(aminomethyl)nicotinate analogue 1a. The cedrenyl ester 1l and the menthoxyisopropyl ester 1a(2') proved most efficacious in all three tumor cell lines. The DNA binding of complexes 1 was assessed by electrophoretic band-shift experiments and found correlated to the terpene structure but not to the observed antiproliferative activities.     

Binding of distamycin A and netropsin to the 12mer DNA duplexes containing mixed AT.GC sequences with at most five or three successive AT base pairs

Circular dichroism (CD), isothermal calorimetric titrations (ITC), and temperature-dependent UV spectroscopy were used to investigate binding of the minor groove-directed ligands distamycin A (Dst) and netropsin (Net) to the following duplexes: d(GTTAGTATTTGG). d(CCAAATACTAAC), d(GTTAGTATATGG).d(CCATATACTAAC), d(GTTAGTACTTGG). d(CCAAGTACTAAC), and d(GTTAGTAGTTGG).d(CCAACTACTAAC). Our results reveal that Dst binds within the minor grooves of these dodecamers that contain five-AT and/or four-AT.GC binding sites exclusively in a dimeric high-affinity 2:1 binding mode (K approximately 10(16) M(-)(2)). By contrast, Net exhibits high-affinity binding only when it binds in a 1:1 mode (K(1) approximately 10(9) M(-)(1)) to the two duplexes that contain five-AT sites (5'-TATTT-3' and 5'-TATAT-3'). Its further binding to these two duplexes occurs in a low-affinity mode (K(2) approximately 10(6) M(-)(1)) and results in the formation of 2:1 Net-DNA complexes. To the other two duplexes that contain sequences with at most three AT consecutive base pairs Net binds in two distinctive low-affinity 1:1 binding modes (K(1) approximately 10(7) M(-)(1), K(2) approximately 10(6) M(-)(1)). Competition experiments (CD and ITC titrations) reveal that Dst entirely displaces Net from its 1:1 and 2:1 complexes with any of the four duplexes. We discuss and interpret our optical and calorimetric results in the context of the available structural information about the complexes between DNA and the sequence-specific minor groove binders Dst and Net.     

DNA-binding and photoactivated enantiospecific cleavage of chiral polypyridyl ruthenium(II) complexes

A series of enantiomeric polypyridyl ruthenium(II) complexes Delta- and Lambda-[Ru(bpy)2CNOIP](PF6)2 (Delta-1 and Lambda-1; BPY=2,2'-bipyridine, CNOIP=2-(2-chloro-5-nitrophenyl)imidazo[4,5-f][1,10]phenanthroline), Delta- and Lambda-[Ru(bpy)2HPIP](PF6)2 (Delta-2 and Lambda-2; HPIP=2-(2-hydroxyphenyl)imidazo[4,5-f][1,10]phenanthroline), Delta- and Lambda-[Ru(bpy)2DPPZ](PF6)2 (Delta-3 and Lambda-3; DPPZ=dipyrido[3,2:a-2',3':c]-phenazine), Delta- and Lambda-[Ru(bpy)2TAPTP](PF6)2 (Delta-4 and Lambda-4; TAPTP=4,5,9,18-tetraazaphenanthreno[9,10-b] triphenylene) have been synthesized. Binding of these chiral complexes to calf thymus DNA has been studied by spectroscopic methods, viscosity, and equilibrium dialysis. The experimental results indicated that all the enantiomers of these complexes bound to DNA through an intercalative mode, but the binding affinity of each chiral complex to DNA was different due to the different shape and planarity of the intercalative ligand. After binding to DNA, the luminescence property of complex 1 was distinctly different from complexes 2 to 4. Upon irradiation at 302 nm, complexes 2-4 were found to promote the cleavage of plasmid pBR 322 DNA from supercoiled form I to nicked form II, and obvious enantioselectively was observed on DNA cleavage for the enantiomers of complexes 2 and 4. The mechanisms for DNA cleavage by these enantiomeric complexes were also proposed.     

DNA condensation by polyamines: a laser light scattering study of structural effects.

Polyamines such as spermidine and spermine are abundant in living cells and are believed to aid in the dense packaging of cellular DNA. DNA condensation is a prerequisite for the transport of gene vectors in living cells. To elucidate the structural features of polyamines governing DNA condensation, we studied the collapse of lambda-DNA by spermine and a series of its homologues, H2N(CH2)3NH(CH2)n=2-12NH(CH2)3NH2 (n = 4 for spermine), using static and dynamic light scattering techniques. All polyamines provoked DNA condensation; however, their efficacy varied with the structural geometry of the polyamine. In 10 mM sodium cacodylate buffer, the EC50 values for DNA condensation were comparable (4 +/- 1 microM) for spermine homologues with n = 4-8, whereas the lower and higher homologues provoked DNA condensation at higher EC50 values. The EC50 values increased with an increase in the monovalent ion (Na+) concentration in the buffer. The slope of a plot of log [EC50(polyamine4+)] against log [Na+] was approximately 1.5 for polyamines with even number values of n, whereas the slope value was approximately 1 for compounds with odd number values of n. Dynamic light scattering measurements showed the presence of compact particles with hydrodynamic radii (Rh) of about 40-50 nm for compounds with n = 3-6. Rh increased with further increase in methylene chain length separating the secondary amino groups of the polyamines (Rh = 60-70 nm for n = 7-10 and >100 nm for n = 11 and 12). Determination of the relative binding affinity of polyamines to DNA using an ethidium bromide displacement assay showed that homologues with n = 2 and 3 as well as those with n > 7 had significantly lower DNA binding affinity compared to spermine and homologues with n = 5 and 6. These data suggest that the chemical structure of isovalent polyamines exerts a profound influence on their ability to recognize and condense DNA, and on the size of the DNA condensates formed in aqueous solution.     

Energetics of DNA end binding by E.coli RecBC and RecBCD helicases indicate loop formation in the 3'-single-stranded DNA tail

We examined the equilibrium binding of Escherichia coli RecBC and RecBCD helicases to duplex DNA ends possessing pre-existing single-stranded (ss) DNA ((dT)(n)) tails varying in length (n=0 to 20 nucleotides) in order to determine the contributions of both the 3' and 5' single strands to the energetics of complex formation. Protein binding was monitored by the fluorescence enhancement of a reference DNA labeled at its end with a Cy3 fluorophore. Binding to unlabeled DNA was examined by competition titrations with the Cy3-labeled reference DNA. The affinities of both RecBC and RecBCD increase as the 3'-(dT)(n) tail length increases from zero to six nucleotides, but then decrease dramatically as the 3'-(dT)(n) tail length increases from six to 20 nucleotides. Isothermal titration calorimetry experiments with RecBC show that the binding enthalpy is negative and increases in magnitude with increasing 3'-(dT)(n) tail length up to n=6 nucleotides, but remains constant for n > or =6. Hence, the decrease in binding affinity for 3'-(dT)(n) tail lengths with n > or =6 is due to an unfavorable entropic contribution. RecBC binds optimally to duplex DNA with (dT)6 tails on both the 3' and 5'-ends while RecBCD prefers duplex DNA with 3'-(dT)6 and 5'-(dT)10 tails. These data suggest that both RecBC and RecBCD helicases can destabilize or "melt out" six base-pairs upon binding to a blunt DNA duplex end in the absence of ATP. These results also provide the first evidence that a loop in the 3'-ssDNA tail can form upon binding of RecBC or RecBCD with DNA duplexes containing a pre-formed 3'-ssDNA tail with n > or =6 nucleotides. Such loops may be representative of those hypothesized to form upon interaction of a Chi site contained within the unwound 3' ss-DNA tail with the RecC subunit during DNA unwinding.     

Interaction of minor groove binding ligands with long AT tracts.

We have used quantitative DNase I footprinting to examine the ability of distamycin and Hoechst 33258 to discriminate between different arrangements of AT residues, using synthetic DNA fragments containing multiple blocks of (A/T)6or (A/T)10in identical sequence environments. Previous studies have shown that these ligands bind less well to (A/T)4sites containing TpA steps. We find that in (A/T)6tracts distamycin shows little discrimination between the various sites, binding approximately 2-fold stronger to TAATTA than (TA)3, T3A3and GAATTC. In contrast, Hoechst 33258 binds approximately 20-fold more tightly to GAATTC and TAATTA than T3A3and (TA)3. Hydroxyl radical footprinting reveals that both ligands bind in similar locations at the centre of each AT tract. At (A/T)10sites distamycin binds with similar affinity to T5A5, (TA)5and AATT, though bands in the centre of (TA)5are protected at approximately 50-fold lower concentration than those towards the edges. Hoechst 33258 shows a similar pattern of preference, with strong binding to AATT, T5A5and the centre of (TA)5. Hydroxyl radical footprinting reveals that at low concentrations both ligands bind at the centre of (TA)5and A5T5, while at higher concentrations ligand molecules bind to each end of the (A/T)10tracts. At T5A5two ligand molecules bind at either end of the site, even at the lowest ligand concentration, consistent with the suggestion that these compounds avoid the TpA step. Similar DNase I footprinting experiments with a DNA fragment containing T n (n = 3-6) tracts reveals that both ligands bind in the order T3< T4 << T5 = T6.     

Alternative heterocycles for DNA recognition: a 3-pyrazole/pyrrole pair specifies for G.C base pairs

Synthetic ligands comprising three aromatic amino acids, pyrrole (Py), imidazole (Im), and hydroxypyrrole (Hp), specifically recognize predetermined sequences as side-by-side pairs in the minor groove of DNA. To expand the repertoire of aromatic rings that may be utilized for minor groove recognition, three five-membered heterocyclic rings, 3-pyrazolecarboxylic acid (3-Pz), 4-pyrazolecarboxylic acid (4-Pz), and furan-2-carboxylic acid (Fr), were examined at the N-terminus of eight-ring hairpin polyamide ligands. The DNA binding properties of 3-Pz, 4-Pz, and Fr each paired with Py were studied by quantitative DNase I footprinting titrations on a 283 bp DNA restriction fragment containing four 6-bp binding sites 5'-ATNCCTAA-3' (N = G, C, A, or T; 6-bp polyamide binding site is underlined). The pair 3-Pz/Py has increased binding affinity and sequence specificity for G.C bp compared with Im/Py.     

Syntheses,characterization and DNA-binding studies of ruthenium(II) terpyridine complexes: [Ru(tpy)(PHBI)]2+ and [Ru(tpy)(PHNI)]2+

Two novel tridentate ligands, 2-(2-benzimidazole)-1,10-phenanthroline (PHBI) and 2-(2-naphthoimidazole)-1,10-phenanthroline (PHNI), and their heteroleptic complexes [Ru(tpy)(PHBI)](ClO(4))(2).2H(2)O (1) and [Ru(tpy)(PHNI)](ClO(4))(2).H(2)O (2) (tpy=2,2':6',2"-terpyridyl) have been synthesized and characterized by elemental analysis, mass spectra, 1H NMR, and electronic spectroscopy. The electrochemical behaviors of the two novel complexes were studied by cyclic voltammetry. The DNA-binding properties of the two complexes were investigated by spectroscopic methods and viscosity measurements. The results indicated that the two complexes interact with DNA in different binding modes. Complex 1 may bind to DNA via electrostatic interaction, while complex 2 binds to DNA by partial intercalation via the extended naphthyl ring into the base pairs of DNA.     

Binding kinetics and ligand specificity for the interactions of the C2B domain of synaptogmin II with inositol polyphosphates and phosphoinositides

Synaptotagmin II (Syt II) is a key protein in the calcium-dependent exocytosis of synaptic vesicles. It contains two domains homologous to the C2 regulatory region of protein kinase C. The C2A domain acts as a calcium sensor, while the C2B domain has high affinity for inositol polyphosphates (InsP(n)()s) and phosphoinositide polyphosphates (PtdInsP(n)()s). We describe the use of a surface plasmon resonance biosensor in determining the binding kinetics of the C2B domain with InsP(n)() and PtdInsP(n) ligands. Biosensor surfaces were prepared with covalently attached Ins(1,4,5)P(3), Ins(1,3,4,5)P(4), and InsP(6) ligands. The interactions of bacterially expressed His(6)-tagged C2B and (C2A+C2B) domains of Syt II were examined in the presence and absence of competing InsP(n)s and PtdInsP(n)s. Both His(6)-C2B and His(6)-(C2A+C2B) exhibited the highest affinity for the Ins(1,3,4,5)P(4)-modified surface with a K(D) value of 6 nM. The His(6)-(C2A+C2B) had a 10-fold lower association rate constant for the InsP(6)-linked surface (k(a) = 4.6 x 10(3) M(-1) s(-1)) than for the Ins(1,3,4,5)P(4)-modified surface (k(a) = 6.8 x 10(4) M(-1) s(-1)). Two water-soluble phosphoinositides, dioctanoyl-PtdIns(3,4,5)P(3) and dioctanoyl-PtdIns(4,5)P(2), were superior to the soluble InsP(n)s in displacing binding to the Ins(1,3,4,5)P(4)-modified surface. The binding of His(6)-C2B and His(6)-(C2A+C2B) to InsP(n) surfaces did not show significant calcium dependence. These data support a model in which the binding of the C2B domain of Syt II to PtdInsP(n)s is important for the docking and/or fusion of the secretory vesicles to the synaptic plasma membrane.     

Synthesis, crystal structure, cytotoxic activity and DNA-binding properties of the copper (II) and zinc (II) complexes with 1-[3-(2-pyridyl)pyrazol-1-ylmethyl]naphthalene

A new ligand L, 1-[3-(2-pyridyl)pyrazol-1-ylmethyl]naphthalene, and its two metal complexes, [Cu(L)3](ClO4)2 (1) and [Zn(L)3](ClO4)2(H2O)2 (2), have been synthesized and characterized. The crystal structure of complex 1 was determined by single crystal X-ray diffraction, which crystallized in monoclinic, space group P2(1)/n with unit cell parameters, a = 12.710(4) angstroms, b = 12.135(3) angstroms, c = 33.450(9) angstroms, beta = 93.281(5) degrees and Z = 4. The Cu atom was six-coordinated to N(1), N(2), N(4), N(5), N(7) and N(8) from three L ligands and formed a slightly distorted octahedral geometry. Complexes 1 and 2, and ligand L were subjected to biological tests in vitro using three different cancer cell lines (HL-60, BGC-823 and MDA-MB-435). Complex 1 showed significant cytotoxic activity against three cancer cell lines. The interactions of complexes 1 and 2, and ligand L with calf thymus DNA were then investigated by thermal denaturation, viscosity measurements and spectrophotometric methods. The experimental results indicated that complexes 1 and 2 bound to DNA by intercalative mode via the ligand L. The intrinsic binding constants of complexes 1 and 2, and ligand L with DNA were 1.8 x 10(4), 5.4 x 10(3) and 2.76 x 10(3) M(-1), respectively.     

Binding and repair of O6-ethylguanine in double-stranded oligodeoxynucleotides by recombinant human O6-alkylguanine-DNA alkyltransferase do not exhibit significant dependence on sequence context.

Double-stranded (ds) oligodeoxynucleotides (29mers) containing an O6-ethylguanine (O6-EtGua) flanked 5' and 3' by different bases (5'..TGT..3'; 5'..CGG..3', 5'..GGT..3'; 5'..GGG..3'; 5'..GGA..3') were synthesized to investigate the binding and repair characteristics of recombinant human O6-alkylguanine-DNA alkyltransferase (AT) in vitro. The apparent association constant (KA(app)) of AT to the oligomers and the repair rate constant for O6-EtGua (k) respectively, were determined by gel retardation and a monoclonal antibody-based filter binding assay. When ds- or single-stranded (ss) oligomers with or without O6-EtGua were used, no major differences in KA(app) values were observed with either substrate: KA(app) values for native AT were 7.1 and 8.4 x 10(5) M(-1) respectively, for unmodified and [O6-EtGua]-containing ds-oligomers. The corresponding values for ss-oligomers were 1.0 and 4.9 x 10(5) M(-1). The N-terminal first 56 amino acids of AT only exert a limited influence on DNA binding; the KA(app) values for an N-terminally truncated AT protein (1.1 x 10(5) M(-1)) and native AT were of the same order. Moreover, KA(app) was hardly affected by Cys(145)-methylated AT (2.0 x 10(5) M(-1)). The k-values (6.5-11.5 x 10(6) M(-1)s(-1)) were not significantly dependent on nucleotide sequence. k-values of 5.3 and 4.0 x 10(6) M(-1)s(-1) respectively, were obtained with the N-terminally truncated AT protein and for repair of the postreplicative mispair [O6-EtGua]: T by native AT. The low KA(app), the negligible influence on O6 of ethylation, and the minor modulation KA(app) and k by varying the bases flanking O6-EtGua, all indicate that the binding of AT to DNA is non-specific and mediated mainly by ionic interactions [reduced KA(app) and k-values at increased ionic strength]. Surplus DNA reduces the rate of O6-EtGua repair in ds-oligomers by competitive binding of AT molecules. The reaction mechanism of AT with DNA in vivo requires further investigation.     

Studies of the cellulolytic system of Trichoderma reesei QM 9414. Reaction specificity and thermodynamics of interactions of small substrates and ligands with the 1,4-beta-glucan cellobiohydrolase II

The 1,4-beta-glucan cellobiohydrolase II (CBH II) from Trichoderma reesei QM 9414 catalyses the hydrolysis of the 4-methylumbelliferyl beta-D-glycosides derived from cellotriose, cellotetraose and cellopentaose [MeUmb(Glc)n; n = 3 - 5]. The reaction has been followed by quantitative high-performance liquid chromatography. Specific activity for cellobiose removal at apparent substrate saturation were determined as (0.8 +/- 0.2) min-1 for MeUmb(Glc)3 and (9 +/- 2) min-1 for MeUmb(Glc)4. The enzyme showed a deviant specificity with MeUmb(Glc)5 as substrate. Two chromophoric products were formed simultaneously [MeUmb(Glc)3 and MeUmb(Glc)2] with turn-over numbers (17 +/- 4) min-1 and (21 +/- 6) min-1, respectively. Methylumbelliferyl beta-glucoside (MeUmbGlc) and the corresponding cellobioside [MeUmb(Glc)2] were used in equilibrium binding experiments. Both ligands yielded one binding site per molecule of Mr = 54000 upon forced flow dialysis (diafiltration). The association constants found were in fair agreement with those determined from MeUmb fluorescence quenching titrations. Quenching was total at all temperatures investigated for MeUmb(Glc)2, whereas for MeUmbGlc it increased from 80% to 100% between 2 degrees C and 20 degrees C. The association constants fitted linear van't Hoff plots in both cases. MeUmb(Glc)2 and MeUmbGlc were also used as indicator ligands to determine the association constants and thermodynamic parameters of several non-chromophoric ligands of CBH II. The binding of glucose increased the affinity for MeUmb(Glc)2 whereas it displaced MeUmbGlc from its complex. A putative binding site of the CBH II containing four subsites can be proposed. The thermodynamic data for methyl beta-D-glucopyranoside and cellobiose as ligands also point at an extended binding site.     

Cyclic cytidine monophosphate stimulates DNA synthesis by bovine mammary tissue in vitro

Mammary gland biopsies were taken from midpregnant heifers (n = 4), cut into pieces .5 mm thick and 3 - 5 mm2 and incubated for 48 hours in Eagle's Minimum Essential Medium containing 0, .1 or 1 micrograms/ml insulin and 0, 10(-8), 10(-7), 10(-6), 10(-5), or 10(-4) M dibutyryl cyclic 3', 5', cytidine monophosphate (dbcCMP). With 0 or .1 microgram/ml insulin, dbcCMP decreased incorporation of tritiated thymidine into DNA. Similar declines in DNA synthesis were observed with sodium butyrate, suggesting that the decline was due to the butyrate rather than to a cyclic CMP-specific effect. With 1 micrograms/ml insulin, dbcCMP increased DNA synthesis. Higher levels of dbcCMP reduced DNA synthesis relative to 10(-6)M dbcCMP, as did sodium butyrate. Thus cCMP is capable of stimulating mammary growth.     

Purification of a HeLa cell nuclear protein that binds selectively to DNA irradiated with ultra-violet light.

Ultraviolet (UV) light induces a variety of lesions in DNA of which the pyrimidine dimer represents the major species. Pyrimidine dimers exist as both a cyclobutane type and a 6-4' (pyrimidine-2'-one) photoproduct. We have purified a protein of M(r) approximately 125,000 from HeLa cell nuclei which binds efficiently to double-stranded DNA irradiated with UV light but not to undamaged DNA. This protein was designated UVBP1 (UV damage binding protein 1). UVBP1 did not recognise DNA damaged by cisplatin. Using oligonucleotides with a single dipyrimidine site for induction of UV photoproducts, binding of UVBP1 to a TC-containing substrate was shown to be more efficient than to substrates containing a TT, a CT or a CC pair. This binding specificity implies selective recognition of the 6-4' photoproduct. Further evidence for this was provided by the finding that hot alkali treatment of the substrate (which selectively hydrolyses 6-4' photoproducts) abrogated binding of UVBP1, whereas incubation with DNA photolyase to remove cyclobutane dimers did not. No detectable DNA helicase, ATPase or exonuclease activity was associated with the purified protein. We suggest that UVBP1 may be involved in the lesion recognition step of DNA excision repair and could contribute to the preferential repair of 6-4' photoproducts from the DNA of UV-irradiated mammalian cells.