Neil P. Johnson

Metal Antitumor Compounds: The Mechanism of Action of Platinum Complexes

Cisplatin (cis-diamminedichloroplatinum(II)) is widely used in the treatment of testicular and ovarian cancers. A number of biological and biochemical results indicate that the reaction of cisplatin with DNA is responsible for the cytotoxic action of this drug. The effect of platinum compounds on the conformation and stability of DNA has been investigated and several platinum-DNA adducts have been identified in vitro and in vivo. Preliminary experiments have quantified the effect of these different lesions on DNA replication, their capacity to induce mutations and their susceptibility to DNA repair processes. Additional DNA damage may be created by platinum(IV) compounds, perhaps during their reduction to platinum(II) compounds by the cell.

Biophysical studies of the modification of DNA by antitumour platinum coordination complexes

Cisplatin (cis-diamminedichloroplatinum(II] is widely used in the treatment of various human tumours. A large body of experimental evidence indicates that the reaction of cisplatin with DNA is responsible for the cytostatic action of this drug. Several platinum-DNA adducts have been identified and their effect on the conformation of DNA has been investigated. Structural studies of platinum-DNA adducts now permit a reasonably good explanation of the biophysical properties of platinated DNA. Antitumouractive platinum compounds induce in DNA, at low levels of binding, local conformational alterations which have the character of non-denaturing distortions. It is likely that these changes occur in DNA due to the formation of intrastrand cross-links between two adjacent purine residues. On the other hand, the modification of DNA by antitumour-inactive complexes results in the formation of more severe local denaturation changes. Conformational alterations induced in DNA by antitumour-active platinum compounds may be reparable with greater difficulty than those induced by the inactive complexes. Alternatively, non-denaturation change induced in DNA by antitumour platinum drugs could represent more significant steric hindrance against DNA replication as compared with inactive complexes.

Preparation and interconversion of dimeric di-µ-hydroxo and tri-µ-hydroxo complexes of platinum(II) and palladium(II) with 2,2′-bipyridine and 1,10-phenanthroline

Treatment of [Pt(L)I2][L = 2,2′-bipyridine (bipy) or 1,10-phenanthroline (phen)] with AgNO3 in acetone gives the nitrato complexes [PtL(ONO2)2]. The palladium analogues were prepared from [Pd(L)Cl2] in dilute nitric acid. Dissolution of [ML(ONO2)2](M = Pd or Pt) in water results in the formation of the hydroxo-bridged dimers [LM(µ-OH)2ML][NO3]2 plus nitric acid. Reaction of [M(L)Cl2] with AgNO3 in water gives [LM(µ-OH)2ML][NO3]2 directly as the sole product. The dimers are resistant to substitution, although prolonged heating in aqueous nitric acid reforms [ML(ONO2)2]. The dimers add 1 mol of OH– to form the very stable trihydroxo-bridged compounds [LM(µ-OH)3ML]+(M = Pt, deep red; M = Pd, deep yellow) where each metal is five-co-ordinate. These complexes are slowly cleaved by hydroxide to give [ML(OH)2], which was also prepared either by base hydrolysis or by reaction of [M(L)Cl2] with Ag2O. Addition of HX (X = NO3, or ClO4) to [PtL(OH)2] affords [LPt(µ-OH)3PtL]+, [LPt(µ-OH)2PtL]2+ or [PtL(ONO2)2] at pH 8, 4, and 1 respectively. The complexes have been characterised by i.r., u.v., and n.m.r. (195Pt, 13C, and 1H) spectroscopy.

Spectroscopic studies of position-specific DNA “breathing” fluctuations at replication forks and primer-template junctions

Junctions between ssDNA and dsDNA sequences are important in many cellular processes, including DNA replication, transcription, recombination, and repair. Significant transient conformational fluctuations (“DNA breathing”) can occur at these ssDNA–dsDNA junctions. The involvement of such breathing in the mechanisms of macromolecular complexes that operate at these loci is not well understood, in part because these fluctuations have been difficult to measure in a position-specific manner. To address this issue we constructed forked or primer-template DNA constructs with 1 or 2 adjacent 2-aminopurine (2-AP) nucleotide residues (adenine analogues) placed at specific positions on both sides of the ssDNA–dsDNA junction. Unlike canonical DNA bases, 2-AP absorbs, fluoresces, and displays CD spectra at wavelengths >300 nm, where other nucleic acid and protein components are transparent. We used CD and fluorescence spectra and acrylamide quenching of these probes to monitor the extent and nature of DNA breathing of A-T base pairs at specific positions around the ssDNA–dsDNA junction. As expected, spectroscopically measurable unwinding penetrates ≈2 bp into the duplex region of these junctions under physiological conditions for the constructs examined. Surprisingly, we found that 2-AP bases at ssDNA sites directly adjacent to ssDNA–dsDNA junctions are significantly more unstacked than those at more distant ssDNA positions. These local and transient DNA conformations on both sides of ssDNA–dsDNA junctions may serve as specific interaction targets for enzymes that manipulate DNA in the processes of gene expression.

Solution conformation of 2-aminopurine dinucleotide determined by ultraviolet two-dimensional fluorescence spectroscopy

We have observed the conformation-dependent electronic coupling between the monomeric subunits of a dinucleotide of 2-aminopurine (2-AP), a fluorescent analog of the nucleic acid base adenine. This was accomplished by extending two-dimensional fluorescence spectroscopy (2D FS) - a fluorescence-detected variation of 2D electronic spectroscopy - to excite molecular transitions in the ultraviolet (UV) regime. A collinear sequence of four ultrafast laser pulses centered at 323 nm was used to resonantly excite the coupled transitions of 2-AP dinucleotide. The phases of the optical pulses were continuously swept at kilohertz frequencies, and the ensuing nonlinear fluorescence was phase-synchronously detected at 370 nm. Upon optimization of a point-dipole coupling model to our data, we found that in aqueous buffer the 2-AP dinucleotide adopts an average conformation in which the purine bases are non-helically stacked (center-to-center distance R12 = 3.5 Å ± 0.5 Å, twist angle θ12 = 5° ± 5°), which differs from the conformation of such adjacent bases in duplex DNA. These experiments establish UV-2D FS as a method for examining the local conformations of an adjacent pair of fluorescent nucleotides substituted into specific DNA or RNA constructs, which will serve as a powerful probe to interpret, in structural terms, biologically significant local conformational changes within the nucleic acid framework of protein-nucleic acid complexes.

DNA conformational changes at the primer-template junction regulate the fidelity of replication by DNA polymerase

Local conformational changes in primer-template (P/T) DNA are involved in the selective incorporation of dNTP by DNA polymerases (DNAP). Here we use near UV CD and fluorescence spectra of pairs of base analogue probes, substituted either at the primer terminus or in the coding region of the template strand, to monitor and interpret conformational changes at and near the coding base of the template in P/T DNA complexes with Klenow fragment (KF) DNAP as the polymerase moves through the nucleotide addition cycle. Incoming dNTPs and rNTPs encounter binary complexes in which the 3′-end of the primer shuttles between the polymerization (pol) and exonuclease (exo) sites of DNAPs, even for perfectly complementary P/T DNA sequences. We have used spectral changes of probes inserted in both strands to monitor this two-state distribution and determine how it depends on the formation of ternary complexes with both complementary (“correct”) and noncomplementary (“incorrect”) NTPs and on the local sequence of the P/T DNA. The results show that the relative occupancy of the exo and pol sites is coupled to conformational changes in the P/T DNA of the complex that are partially regulated by the incoming NTP. We find that the coding base on the template strand is unperturbed by the binding of incorrect dNTPs, while binding of complementary rNTPs induces a novel template conformation. We conclude that, in addition to its editing function, primer strand occupancy of the 3′-exo site may also serve as a regulatory checkpoint for accurate dNTP selection in DNA synthesis.

Kinetic studies of the hydrolysis of platinum-DNA complexes by nuclease S1.

The antitumor agent cis-diamminedichloroplatinum(II) (cis-DDP) reacts covalently with DNA and disrupts its secondary structure. Damaged DNA, but not native DNA, is readily digested by S1 nuclease, an endonuclease specific for single stranded polynucleotides. We have measured S1 nuclease digestion of platinated DNA by the release of platinum-DNA adducts and compared it with digestion of unplatinated DNA. The rate of hydrolysis of damaged substrate from platinum-DNA complexes was less than the overall rate of digestion of nucleotides. Similar results were observed for platinum-DNA complexes in native, denatured or renatured conformations. The hydrolysis of denatured platinum-DNA complexes, rb = 0.075 platinum per nucleotide, obeyed Michaelis-Menten kinetics. Taking into account the level of DNA damage, Vm, for the release of platinated adducts was 0.6 times smaller than for digestion of unplatinated DNA. Km values and competition experiments indicated that the enzyme bound equally well to platinated and unplatinated substrates. Similar results were obtained for denatured DNA complexes with trans-DDP while [PtCl(diethylenetriamine)]Cl had no influence on nuclease digestion. These results suggest that bifunctional platinum-DNA lesions have contradictory effects on the hydrolysis of double stranded DNA by S1 nuclease. On one hand they create nuclease sensitive substrate by disrupting DNA secondary structure. On the other, they inhibit digestion of the damaged strand by increasing the activation energy for hydrolysis.

Local Conformations and Competitive Binding Affinities of Single- and Double-stranded Primer-Template DNA at the Polymerization and Editing Active Sites of DNA Polymerases

In addition to their capacity for template-directed 5′ → 3′ DNA synthesis at the polymerase (pol) site, DNA polymerases have a separate 3′ → 5′ exonuclease (exo) editing activity that is involved in assuring the fidelity of DNA replication. Upon misincorporation of an incorrect nucleotide residue, the 3′ terminus of the primer strand at the primer-template (P/T) junction is preferentially transferred to the exo site, where the faulty residue is excised, allowing the shortened primer to rebind to the template strand at the pol site and incorporate the correct dNTP. Here we describe the conformational changes that occur in the primer strand as it shuttles between the pol and exo sites of replication-competent Klenow and Klentaq DNA polymerase complexes in solution and use these conformational changes to measure the equilibrium distribution of the primer between these sites for P/T DNA constructs carrying both matched and mismatched primer termini. To this end, we have measured the fluorescence and circular dichroism spectra at wavelengths of >300 nm for conformational probes comprising pairs of 2-aminopurine bases site-specifically replacing adenine bases at various positions in the primer strand of P/T DNA constructs bound to DNA polymerases. Control experiments that compare primer conformations with available x-ray structures confirm the validity of this approach. These distributions and the conformational changes in the P/T DNA that occur during template-directed DNA synthesis in solution illuminate some of the mechanisms used by DNA polymerases to assure the fidelity of DNA synthesis.

Investigation of the Secondary DNA-binding Site of the Bacterial Recombinase RecA

The L2 loop is a DNA-binding site of RecA protein, a recombinase from Eschericha coli. Two DNA-binding sites have been functionally defined in this protein. To determine whether the L2 loop of RecA protein is part of the primary or secondary binding site, we have constructed proteins with site-specific mutations in the loop and investigated their biological, biochemical, and DNA binding properties. The mutation E207Q inhibits DNA repair and homologous recombination in vivo and prevents DNA strand exchange in vitro (Larminat, F., Cazaux, C., Germanier, M., and Defais, M. (1992) J. Bacteriol. 174, 6264–6269; Cazaux, C., Larminat, F., Villani, G., Johnson, N. P., Schnarr, M., and Defais, M. (1994) J. Biol. Chem. 269, 8246–8254). We have found that mutant protein RecAE207Qlacked one of the two single stranded DNA-binding sites of wild type RecA. The remaining site was functional, and biochemical activities of the mutant protein were the same as wild type RecA with ssDNA in the primary binding site. The second mutation, E207K, reduced but did not eliminate DNA repair, SOS induction, and homologous recombinationin vivo. In the presence of ATP, mutant protein RecAE207K catalyzed DNA strand exchange in vitro at a slower rate than wild type protein, and ssDNA binding at site I was competitively inhibited. These results show that the L2 loop is or is part of the functional secondary DNA-binding site of RecA protein.

Determination of cis-trans isomers of amine and pyridine platinum(II) complexes by J(PtH) coupling constants

Abstract The coupling constants 3J(PtH) of [Pt(Xpy)2L2] and [Pt(Xpy)(dmso)I2] complexes (Xpy = pyridine, 4-Me-py, 3,5-diMe-py, 4-Cl- py, 4-CN-py, 4-HOCH2-py, 4-CH3COO-py; L = Cl, I, Br, ONO2) have been recorded. The 3J(PtH) values of the cis complexes are 42 ± 2 Hz, while those of the trans derivatives are 31.5 ± 2.5 Hz. 2J(PtH) of [Pt(XNH2)2L2] complexes (X = CH 3, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl; L = Cl, I, ONO2) also have been recorded. The 2J(PtH) values of the cis complexes are 66.5 ± 1.5 Hz, those of the trans derivatives are 58 ± 2 Hz. These results show that measurement of PtH coupling constants is a rapid method of identifying the cis and the trans isomers of platinum(II) complexes.