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-methoxypsoralen and the A and A 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. INTRODUCTIONThe interactions of double stranded DNA with small as well as with large molecules are often divided into contributions from the deoxyribose phosphate backbone and from the nucleobases themselves, although this segregation of binding/recognition components is not easily made. The main electrostatic component of the binding energy of cationic ligands is generally attributed to the negative phosphates of the DNA backbone, whereas the nucleobases contribute hydrophobic and dispersive bonding components but also considerable electrostatic components in terms of hydrogen and dipolar bonding (1,2). Specific interactions with the nucleobases naturally determine the sequence specificity exhibited by a ligand, for proteins they are also assisted by 'textured' properties such as helical pitch, indirectly determined by the base-sequence (3,4). However, the relative importance of electrostatic and hydrophobic contributions in the recognition/ binding process is far from clear, even for rather simple ligands such as typical intercalators and minor groove binders.We recently prepared a DNA analog termed PNA (peptide nucleic acid) containing an uncharged pseudo-peptide backbone composed of N-(2-aminoethyl)glycine units to which the nucleobases are attached (Figure ...
RecA catalyses homologous recombination in Escherichia coli by promoting pairing of homologous DNA molecules after formation of a helical nucleoprotein filament with single-stranded DNA. The primary reaction of RecA with DNA is generally assumed to be unspecific. We show here, by direct measurement of the interaction enthalpy by means of isothermal titration calorimetry, that the polymerisation of RecA on single-stranded DNA depends on the DNA sequence, with a high exothermic preference for thymine bases. This enthalpic sequence preference of thymines by RecA correlates with faster binding kinetics of RecA to thymine DNA. Furthermore, the enthalpy of interaction between the RecA . DNA filament and a second DNA strand is large only when the added DNA is complementary to the bound DNA in RecA. This result suggests a possibility for a rapid search mechanism by RecA . DNA filaments for homologous DNA molecules.Keywords : RecA ; recombination ; calorimetry ; protein-DNA interaction ; fluorescence.General genetic recombination is a process, common to all forms of life, by which new combinations of genetic material or nucleic acid sequences are generated. RecA is a key component of general genetic recombination in Escherichia coli [I -31, and related proteins with similar functions are found in a variety of organisms [3, 41. Genetic recombination consists of strand exchange between two homologous DNA molecules, and the reaction facilitates post-replicative DNA repair and is important for DNA segregation in cell division. Purified RecA in vitro promotes the strand-exchange reaction in the presence of cofactor ATP [5, 61, and has been extensively studied to understand the mechanism of homologous-recombination reactions (reviewed in [3,7-9]). RecA has a molecular mass of 38 kDa [lo], but it polymerises into very-high molecular-mass filaments. RecA binds co-operatively to any single-stranded DNA [9], and forms nucleofilaments by arranging itself in a helical manner around the DNA [ l l , 121. Each filament can bind, in the presence of a cofactor, a second DNA and thus direct two DNA molecules towards each other in preparation for strand exchange [13-161. Binding of a second DNA molecule only occurs after saturation of the first DNA-binding site in RecA; the stoichiometry is 3 DNA bases/RecA monomer for both interactions (3 base pairs/RecA monomer if the second DNA molecule is a duplex) [15]. Although structural details begin to accumulate, the physical nature of the DNA binding in RecA and the mechanisms for these protein-mediated DNA reactions are still largely unknown.To better understand the mechanism for RecA pairing of two homologous DNA molecules, we have studied the thermodynamics and kinetics of RecA-DNA interactions. Calorimetry directly measures the heat of interaction for a chemical reaction and gives thermodynamic parameters. We show here that iso- MATERIALS AND METHODSMaterials and experimental conditions. RecA was purified as described elsewhere [20] with HPLC (DEAE 5PW, Tosoh) as a final step. The HPLC-elu...
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