Strong DNA Binding Research Articles

Slow diffusion in situ ruthenium/ligand reaction: Crystal structures, fluorescence and biological properties

Promoted by RuCl3·3H2O coordination in the mixed solvent DMF/H2O, diverse in situ S–S bond reactions such as S–S bond scission and S-oxidation occurred in the disulfide ligand of 2,2′-dithiodibenzoic acid (dtdb) to yield the new sulfinato-benzoate ligand (sb). The X-ray analysis of complexes of [Ru(phen)2(sb)] (1) and [Ru(bipy)2(sb)·H2O] (2) revealed that in both complexes, the ruthenium ion was found to be in an octahedral geometry, coordinating to the sulfur atom, rather than the oxygen of sulfinate. The complexes were found to be active against the bacterial strains tested with MIC ranging from 14.3–261μM. In addition, the metal complexes present strong DNA binding affinity constants in the major or minor grooves at the order of magnitude 105-106M−1. The antioxidant activities of the ligand and its metal complexes were investigated through scavenging effects for DPPH in vitro, indicating that the compounds show stronger activities than some standard antioxidants, such as ascorbic and vitamin C.

Spectroscopic Investigations, Anti-bacterial Activities and DNA-interactions of Metal Complexes (Cr(III), Zn(II), Ni(II)) Containing Phendione Ligand

The transition metal complexes [Cr(acac)2(phendione)]Cl, [Zn(acac)2(phendione)], [Ni(acac)2(phendione)], [Zn(phendione)3](ClO4)2 and [Ni(phendione)2](ClO4)2 were synthesized from 1, 10-phenanthroline-5, 6-dione (phendione). The phendione ligands and its metal complexes were characterized by FTIR, UV-Visible spectroscopy, PL study and in some cases by GC-MS. The red emission band of complex [Zn(acac)2(phendione)] was observed by excitation at 250 nm. DNA binding activities of the complexes were studied by DNA Gel Electrophoresis. DNA binding studies of the synthesized compounds showed that the metal complexes are strong DNA binding and cleaving agents. The [Ni(phendione)2] (ClO4)2 complex was the most effective DNA binding and cleaving agent and [Cr(acac)2(phendione)]Cl complex shows relatively lower affinity to the DNA. The metal complexes easily cause DNA degradation or cleavage (after 2.5 h) with genomic DNA rather than plasmid DNA. Antimicrobial activity studies revealed that all metal complexes of phendione showed efficient resistance to bacteria. The [Cr(acac)2(phendione)]Cl complex showed most potential antimicrobial activities against the bacteria Escherichia coli with an annular radius of the inhibitory zone, 4.33.

Enhancing the Structural Diversity and Bioactivity of Natural Products by Combinatorial Modification Exemplified by Total Tanshinones

AbstractTo enhance the structural diversity of tanshinones and provide more derivatives for the biological studies, a one‐pot combinatorial modification strategy was performed on total tanshinones. Six new quinoxlinetanshinones 1–6 were subsequently isolated from the combinatorial modified semi‐synthetic mixture. The structures were elucidated by spectroscopic analysis in combination with single‐crystal X‐ray diffraction. These quinoxlinetanshinones demonstrated strong DNA binding properties. Rapid synthesis of new quinoxlinetanshinones with significant biological activity highlights the great potential of one‐pot combinatorial modification for the diversification of natural products.

Synthesis, characterization and biological evaluation of transition metal complexes derived from N, S bidentate ligands.

Two bidentate NS ligands were synthesized by the condensation reaction of S-2-methylbenzyldithiocarbazate (S2MBDTC) with 2-methoxybenzaldehyde (2MB) and 3-methoxybenzaldehyde (3MB). The ligands were reacted separately with acetates of Cu(II), Ni(II) and Zn(II) yielding 1:2 (metal:ligand) complexes. The metal complexes formed were expected to have a general formula of [M(NS)2] where M = Cu2+, Ni2+, and Zn2+. These compounds were characterized by elemental analysis, molar conductivity, magnetic susceptibility and various spectroscopic techniques. The magnetic susceptibility measurements and spectral results supported the predicted coordination geometry in which the Schiff bases behaved as bidentate NS donor ligands coordinating via the azomethine nitrogen and thiolate sulfur. The molecular structures of the isomeric S2M2MBH (1) and S2M3MBH (2) were established by X-ray crystallography to have very similar l-shaped structures. The Schiff bases and their metal complexes were evaluated for their biological activities against estrogen receptor-positive (MCF-7) and estrogen receptor-negative (MDA-MB-231) breast cancer cell lines. Only the Cu(II) complexes showed marked cytotoxicity against the cancer cell lines. Both Schiff bases and other metal complexes were found to be inactive. In concordance with the cytotoxicity studies, the DNA binding studies indicated that Cu(II) complexes have a strong DNA binding affinity.

Preparation and testing of quaternized chitosan nanoparticles as gene delivery vehicles.

The aim of this study was to synthesize a chitosan (CS) derivative, a quaternary ammonium salt crystal called N-2-hydroxypropyl trimethyl ammonium chloride chitosan (HACC), and test a series of HACC and pEGFP-DNA complexes at different weight ratios for their efficiency of gene delivery into human cells. CS was modified with cationic etherifying agent to obtain the CS derivative. Fourier transform infrared spectra were recorded on KBr pellets with a spectrometer. (1)H nuclear magnetic resonance (NMR) spectra of HACC were obtained using a spectrometer. HACC was subsequently used to prepare HACC/DNA complexes at different weight ratios by coacervation method. The resulting particle size and surface charge were assessed by laser light scattering using a zeta potential analyzer. The HACC/DNA complex formation and DNA protection in the nanoparticle complex was investigated by gel mobility shift assay and DNase I protection assay, respectively. The cytotoxicity of HACC and HACC/DNA nanoparticles was evaluated by MTT assay using (mesenchymal stem cell) MSC lines. The nanoscale structure of the particles was obtained by transmission electron microscope (TEM). The FTIR spectrum of HACC showed the characteristic quaternary ammonium group absorption band at 1475 cm(-1), which indicated the presence of quaternary ammonium group. The successful synthesis of HACC was also confirmed by (1)H NMR spectrum. HACC showed good solubility in water and was electropositive. HACC efficiently packed and protected pEGFP-DNA at a weight ratio of 10. With increased weight ratios, the surface charge of the composite particle increased from negative to positive, the average particle size increased, and HACC nanoparticle had a higher carrying efficiency. The nanoparticles released DNA in two distinct phases, and 55 % was released within the first 20 h of solubilization. The nanoparticles under TEM showed circular or oval shapes. The particles exhibited no cytotoxicity against human cells. No significant difference in gene delivery efficiency was detected between HACC/pEGFP-GDNF and liposome/pEGFP-GDNF complexes (33.8 vs. 34 %, P = 0.363). In this study, HACC was successfully synthesized, and HACC/DNA complex assembled efficiently. HACC showed strong DNA binding affinity and high protection of DNA and was non-cytotoxic to human cells. The particles had appropriate nanostructure, mean diameter, and DNA release time. The results suggest that HACC nanoparticles are a novel tool for efficient and safe gene delivery.

Novel phenyl and pyridyl substituted derivatives of isoindolines: Synthesis, antitumor activity and DNA binding features

Novel phenyl-substituted (3a-3d, 4a, 5, 8a, 8b and 9a) and pyridyl-substituted (3e-3i, 4b, 8c-8e, 9b and 9c) isoindolines were prepared in the reaction of o-phthalaldehyde and corresponding substituted aromatic and heteroaromatic amines by modification of reaction conditions from low to high temperature and from neutral to acidic environment.The antiproliferative activity of chosen substituted isoindolines was assessed on a panel of tumour cell lines and normal human fibroblasts. The majority of tested compounds was active at the highest tested concentrations phenyl-substituted isoindolines 3a and 3b and pyridyl-substituted isoindoline 3g showed a selective effect at micromolar concentrations on HepG2 cell line in comparison with other tested tumour cell lines and normal human fibroblasts. The strongest yet non-selective effect was observed for the pyridyl-substituted isoindoline 8c. These isoindoline derivatives showed diverse mechanism of action on tumour cell death induction as compounds 3a and 8c probably induced mitotic catastrophe while compound 3b induced apoptosis. Indeed, DNA binding properties evidenced that compounds 8a, 8c and 8d bind to DNA as highly potent DNA intercalators. By contrast, compounds 3b, 3e, 3i, 4a and 5 did not target the DNA. At last, the phenyl-substituted compound 8b proved to be a strong DNA binding compound with sequence selective binding and without DNA intercalation profile.

Characterization of the non-specific DNA binding properties of the Adenoviral IVa2 protein

Human Adenovirus (Ad) is a non-enveloped, icosahedral virus with a linear, double-stranded DNA genome. The Ad IVa2 protein is involved in multiple viral processes including viral late gene transcription and virus assembly. Previous studies have shown that IVa2 loads additional viral proteins onto conserved DNA elements within the Ad genome to regulate these viral processes. IVa2 also possesses strong non-specific DNA binding activity, and it is likely it uses this activity to recruit proteins to the conserved DNA elements. Here we have investigated the non-specific DNA binding activity of IVa2 using nitrocellulose/DEAE filter binding and sedimentation equilibrium techniques. We have analyzed our data using the McGhee and Von Hippel approach [1], and find that IVa2 binds with strong, positive nearest-neighbor cooperativity. In addition, we describe how to apply the McGhee and von Hippel approach to directly analyze sedimentation equilibrium data using non-linear least-squares methods. We discuss the implications of these results with respect to current virus assembly mechanisms.

A cell-penetrating peptide analogue, P7, exerts antimicrobial activity against Escherichia coli ATCC25922 via penetrating cell membrane and targeting intracellular DNA

The antibacterial activities and mechanism of a new P7 were investigated in this study. P7 showed antimicrobial activities against five harmful microorganisms which contaminate and spoil food (MIC=4–32μM). Flow cytometry and scanning electron microscopy analyses demonstrated that P7 induced pore-formation on the cell surface and led to morphological changes but did not lyse cell. Confocal fluorescence microscopic observations and flow cytometry analysis expressed that P7 could penetrate the Escherichia coli cell membrane and accumulate in the cytoplasm. Moreover, P7 possessed a strong DNA binding affinity. Further cell cycle analysis and change in gene expression analysis suggested that P7 induced a decreased expression in the genes involved in DNA replication. Up-regulated expression genes encoding DNA damage repair. This study suggests that P7 could be applied as a candidate for the development of new food preservatives as it exerts its antibacterial activities by penetrating cell membranes and targets intracellular DNA.

Synthesis, spectroscopic characterization, pH dependent redox mechanism and DNA binding behavior of chlorohydroxyaniline derivatives

Three new chlorohydroxyanilines were synthesized and characterized. The results revealed these compounds to have strong antioxidant activity and DNA binding propensity.

Fabrication of Novel Reduction‐Sensitive Gene Vectors Based on Three‐Armed Peptides

To address the inherent barriers of gene transfection, two reduction-sensitive branched polypeptides (RBPs) are synthesized and explored as novel non-viral gene vectors. The introduced disulfide linkages in RBPs facilitate glutathione-triggered intracellular gene release and reduce polymer degradation-induced cytotoxicity. Furthermore, the highly branched architecture concurrently realizes multivalency for strong DNA binding and elicits conformational flexibility for tight DNA compacting, which are beneficial for cellular entry. To increase the endosomal escape of plasmid DNA, pH-sensitive histidyl residues are incorporated into RBPs to improve buffer capacity in an acidic environment. In vitro study demonstrates that RBPs can efficiently mediate the DNA transfection and avoid apparent cytotoxicity in HeLa and COS7. The present gene delivery system offers a simple and flexible approach to fabricate microenvironment-specific branched gene vectors for gene therapy.

Interaction of cholinesterase modulators with DNA and their cytotoxic activity

This research was focused on a study of the binding properties of a series of cholinesterase reactivators compounds K075 (1), K027 (2) and inhibitors compounds K524, K009 and 7-MEOTA (3–5) with calf thymus DNA. The nature of the interactions between compounds 1–5 and DNA were studied using spectroscopic techniques (UV–vis, fluorescence spectroscopy and circular dichroism). The binding constants for complexes of cholinesterase modulators with DNA were determined from UV–vis spectroscopic titrations (K=0.5×104–8.9×105M−1). The ability of the prepared analogues to relax topoisomerase I was studied with electrophoretic techniques and it was proved that ligands 4 and 5 inhibited this enzyme at a concentration of 30μM. The biological activity of the novel compounds was assessed through an examination of changes in cell cycle distribution, mitochondrial membrane potential and cellular viability. Inhibitors 3–5 exhibited a cytotoxic effect on HL-60 (human acute promyelocytic leukaemia) cell culture, demonstrated a tendency to affect mitochondrial physiology and viability, and also forced cells to accumulate in the G1/G0-phase of the cell cycle. The cholinesterase reactivators 1 and 2 were found relatively save from the point of view of DNA binding, whereas cholinesterase inhibitors 3–5 resulted as strong DNA binding agents that limit their plausible use.

Conjugation of benzylvanillin and benzimidazole structure improves DNA binding with enhanced antileukemic properties.

Benzyl-o-vanillin and benzimidazole nucleus serve as important pharmacophore in drug discovery. The benzyl vanillin (2-(benzyloxy)-3-methoxybenzaldehyde) compound shows anti-proliferative activity in HL60 leukemia cancer cells and can effect cell cycle progression at G2/M phase. Its apoptosis activity was due to disruption of mitochondrial functioning. In this study, we have studied a series of compounds consisting of benzyl vanillin and benzimidazole structures. We hypothesize that by fusing these two structures we can produce compounds that have better anticancer activity with improved specificity particularly towards the leukemia cell line. Here we explored the anticancer activity of three compounds namely 2-(2-benzyloxy-3-methoxyphenyl)-1H-benzimidazole, 2MP, N-1-(2-benzyloxy-3-methoxybenzyl)-2-(2-benzyloxy-3-methoxyphenyl)-1H-benzimidazole, 2XP, and (R) and (S)-1-(2-benzyloxy-3-methoxyphenyl)-2, 2, 2-trichloroethyl benzenesulfonate, 3BS and compared their activity to 2-benzyloxy-3-methoxybenzaldehyde, (Bn1), the parent compound. 2XP and 3BS induces cell death of U937 leukemic cell line through DNA fragmentation that lead to the intrinsic caspase 9 activation. DNA binding study primarily by the equilibrium binding titration assay followed by the Viscosity study reveal the DNA binding through groove region with intrinsic binding constant 7.39 µM/bp and 6.86 µM/bp for 3BS and 2XP respectively. 2XP and 3BS showed strong DNA binding activity by the UV titration method with the computational drug modeling showed that both 2XP and 3BS failed to form any electrostatic linkages except via hydrophobic interaction through the minor groove region of the nucleic acid. The benzylvanillin alone (Bn1) has weak anticancer activity even after it was combined with the benzimidazole (2MP), but after addition of another benzylvanillin structure (2XP), stronger activity was observed. Also, the combination of benzylvanillin with benzenesulfonate (3BS) significantly improved the anticancer activity of Bn1. The present study provides a new insight of benzyl vanillin derivatives as potential anti-leukemic agent.

Synthesis, cytotoxicity and DNA binding of oxoazabenzo[de]anthracenes derivatives in colon cancer Caco-2 cells

New oxoazabenzo[de]anthracenes derivatives were synthesised and characterised. Their interactions with calf thymus DNA were studied by UV spectrophotometric analysis and a competitive ethidium bromide displacement assay. Cytotoxicity was determined by MTT assay, against colon adenocarcinoma (Caco-2 cells). Among all the oxoazabenzo[de]anthracenes derivatives reported herein only the piperidino derivative exhibited strong DNA binding properties and cytotoxic activity with IC50 values in the range of 16 ± 1.5 μM (72-h treatment). In addition, the piperidino derivative did not directly inhibit topoisomerase I and topoisomerase II enzymes. The results confirm that the presence of the oxoazabenzo[de]anthracenes together with the piperidino functionality is crucial in exerting DNA binding and cytotoxic properties, hence demonstrating promise as a chemical scaffold for further development of new anticancer agents.

Discovery of indeno[1,2- $$c$$ c ]quinoline derivatives as dual topoisomerases I/II inhibitors: Part 3

(E) -6-Hydroxy-9-methoxy-6-(piperazin-1-yl)-11H -indeno[1,2-c ]quinolin-11-one O-2-(pyrrolidin-1-yl)ethyl oxime (2c) was identified as a potential dual topo I/II inhibitor in our previous paper. In continuation for the search of more potent compounds, we describe herein the preparation of certain indeno[1,2-c ]quinoline derivatives and evaluation of their antiproliferation, DNA binding affinity, and topoisomerases (topo I and topo II) inhibitory activities. Among them, (E) -9-[3-(dimethylamino)propoxy]-11H -indeno[1,2-c ]quinolin-11-one O-3-(dimethylamino)propyl oxime (11b) and its analog 11c exhibited approximately equal activity to the lead compound 2c against the growth of HeLa and A549 cancer cells. Both compounds 11b and 11c were more active than 2c in the inhibition of topo I and topo II. However, none of them exhibited significant DNA binding affinity while 2c was a very strong DNA binding agent. Compound 11b exhibited a high oral bioavailability of 39.8 % while the oral bioavailability of 2c and 11c was only 10.9 and 8.6 %, respectively. The in vivo anti-tumor evaluation of 11b in nude mice bearing subcutaneous breast cancer tumors revealed that treatment with low (10 mg/kg) or high (30 mg/kg) doses of 11b dramatically diminished tumor growth. Therefore, compound 11b is identified as a potential non-DNA intercalating dual topo I/II inhibitor.

Oligoamines grafted hyperbranched polyether as high efficient and serum-tolerant gene vectors

To develop low toxic, high efficient, and excellent serum-tolerant polycation gene delivery systems, a series of oligoamines grafted hyperbranched polyether (oligoamines-g-HBP) were synthesized by conjugating different oligoamines, including triethylenetetramine (TETA) and tetraethylenepentamine (TEPA), onto COOH-functionalized hyperbranched poly(3-ethyl-3-oxetanemethanol). It was found that oligoamines-g-HBP exhibited good buffering capacity, strong DNA binding and high resistance against protein adsorption. In vitro cytotoxicity measurement indicated that oligoamines-g-HBP had much lower cytotoxicity as compared with 25kDa PEI. The transfection efficiency of TEPA-g-HBP/DNA complexes at a certain N/P ratio was significantly higher than that of 25kDa PEI/DNA complexes. Interestingly, it was found that TEPA-g-HBP had much improved serum-tolerant capability as compared with 25kDa PEI even when serum concentration was increased to 30%. Confocal laser images further showed that the amount of YOYO-1 labeled DNA in nuclei got increased with increasing the number of secondary amino ethylene groups in oligoamines-g-HBP. The oligoamines-g-HBP presented great potential as gene delivery vectors for further clinical applications.

Preparation, spectroscopic and thermal characterization of new charge-transfer complexes of ethidium bromide with π-acceptors. In vitro biological activity studies

Ethidium bromide (EtBr) is a strong DNA binder and has been widely used to probe DNA structure in drug-DNA and protein–DNA interaction. Four new charge-transfer (CT) complexes consisting of EtBr as donor and quinol (QL), picric acid (PA), tetracyanoquinodimethane (TCNQ) or dichlorodicyanobenzoquinone (DDQ) as acceptors, were synthesized and characterized by elemental analysis, electronic absorption, spectrophotometric titration, IR, Raman, 1H NMR and X-ray powder diffraction (XRD) techniques. The stoichiometry of these complexes was found to be 1:2 ratio and having the formula [(EtBr)(acceptor)]. The thermal stability of the synthesized CT complexes was investigated using thermogravimetric (TG) analyses, and the morphology and particle size of these complexes were obtained from scanning electron microscopy (SEM). The CT complexes were also tested for its antibacterial activity against two Gram-positive bacteria Staphylococcus aureus and Bacillus subtilis and two Gram-negative bacteria; Escherichia coli and Pseudomonas aeuroginosa strains by using Tetracycline as standard and antifungal property against Aspergillus flavus and Candida albicans by using amphotericin B as standard. The results were compared with the standard drugs and significant conclusions were obtained. The results indicated that the [(EtBr)(QL)2] complex had exerted excellent inhibitory activity against the growth of the tested bacterial strains.

Nuclease Activity of the Human SAMHD1 Protein Implicated in the Aicardi-Goutières Syndrome and HIV-1 Restriction

The human HD domain protein SAMHD1 is implicated in the Aicardi-Goutières autoimmune syndrome and in the restriction of HIV-1 replication in myeloid cells. Recently, this protein has been shown to possess dNTP triphosphatase activity, which is proposed to inhibit HIV-1 replication and the autoimmune response by hydrolyzing cellular dNTPs. Here, we show that the purified full-length human SAMHD1 protein also possesses metal-dependent 3'→5' exonuclease activity against single-stranded DNAs and RNAs in vitro. In double-stranded substrates, this protein preferentially cleaved 3'-overhangs and RNA in blunt-ended DNA/RNA duplexes. Full-length SAMHD1 also exhibited strong DNA and RNA binding to substrates with complex secondary structures. Both nuclease and dNTP triphosphatase activities of SAMHD1 are associated with its HD domain, but the SAM domain is required for maximal activity and nucleic acid binding. The nuclease activity of SAMHD1 could represent an additional mechanism contributing to HIV-1 restriction and suppression of the autoimmune response through direct cleavage of viral and endogenous nucleic acids. In addition, we demonstrated the presence of dGTP triphosphohydrolase and nuclease activities in several microbial HD domain proteins, suggesting that these proteins might contribute to antiviral defense in prokaryotes.

Biological activity and DNA binding studies of 2-substituted benzimidazo[1,2-a]quinolines bearing different amino side chains

This manuscript describes the synthesis and biological activity of 2-substituted benzimidazo[1,2-a]quinolines substituted with different amino side chains on the quinoline nucleus prepared by microwave assisted amination. The majority of compounds were newly synthesized and active at submicromolar IC50 concentrations, while the alkylamino substituents, either acyclic or cyclic, increased antitumor activities in comparison with previously published nitro and amino substituted benzimidazo[1,2-a]quinolines. The compound with the longest tertiary amino side chain (16) was the least active. A series of additional experiments, including DNA binding propensities, topoisomerases I and II inhibition, inhibition of recombinant green fluorescent protein in a cell-free translation system, cell cycle perturbances and cellular localization, was performed to shed more light on the mechanisms of action of the most active compounds. The DNA intercalation activity correlates with anti-proliferative effect. Several DNA intercalators (11, 20 and 21) also evidence some sequence selective DNA binding. However, only N,N-dimethylaminopropyl analogue 11 was unequivocally demonstrated to be a strong DNA-binder and intercalative agent, which efficiently targets DNA in the cells, while the activity of compound 10, with a bulky i-butylamino side chain, points to its potential antimitotic activity.

Quantifying Force-Dependent Binding Kinetics of DNA-Ruthenium Complexes

Ruthenium complexes are small synthetic molecules with a wide range of uses including cancer therapy, and as a research tool to understand chemical carcinogenesis. Specifically, ruthenium dimers like [μ-C4(cpdppz)2(phen)4Ru2]4+ have been engineered to have a high affinity for DNA and a very low dissociation rate. The complex consists of two Ru(phen)2 moieties connected by a flexible linker, and its strong DNA binding inhibits replication of DNA in target cancer cells. To quantify the rate at which DNA binding occurs for this dimer, double-stranded DNA is stretched with optical tweezers, and exposed to the ligand under a fixed applied force. When binding to DNA, the two Ru(phen)2 moieties intercalate between base pairs via a threading mechanism. Intercalation results in an increase in the length of the target DNA, and the rate of this increase depends exponentially upon the applied force. Surprisingly, a fast and a slow binding mode are revealed, indicating multi-step binding.The fast binding rate is weakly correlated with force and is responsible for most of the extension change, while the slow binding rate increases with force. This suggests that intercalation is rapid, and followed by a slower, force-dependent rearrangement of DNA-bound ruthenium complexes. These studies demonstrate the capability of optical tweezers to elucidate the mechanism of complex DNA-ligand interactions, which may facilitate the rational design of DNA binding ligands with specific DNA interaction properties.

Dynamics of bleomycin interaction with a strongly bound hairpin DNA substrate, and implications for cleavage of the bound DNA.

Recent studies involving DNAs bound strongly by bleomycins have documented that such DNAs are degraded by the antitumor antibiotic with characteristics different from those observed when studying the cleavage of randomly chosen DNAs in the presence of excess Fe·BLM. In the present study, surface plasmon resonance has been used to characterize the dynamics of BLM B(2) binding to a strongly bound hairpin DNA, to define the effects of Fe(3+), salt, and temperature on BLM-DNA interaction. One strong primary DNA binding site, and at least one much weaker site, were documented. In contrast, more than one strong cleavage site was found, an observation also made for two other hairpin DNAs. Evidence is presented for BLM equilibration between the stronger and weaker binding sites in a way that renders BLM unavailable to other, less strongly bound DNAs. Thus, enhanced binding to a given site does not necessarily result in increased DNA degradation at that site; i.e., for strongly bound DNAs, the facility of DNA cleavage must involve other parameters in addition to the intrinsic rate of C-4' H atom abstraction from DNA sugars.