Christine Rauch

Daunomycin Intercalation Stabilizes Distinct Backbone Conformations of DNA

Abstract Daunomycin is a widely used antibiotic of the anthracycline family. In the present study we reveal the structural properties and important intercalator-DNA interactions by means of molecular dynamics. As most of the X-ray structures of DNA-daunomycin intercalated complexes are short hexamers or octamers of DNA with two drug molecules per doublehelix we calculated a self complementary 14-mer oligodeoxyribonucleotide duplex d(CGCGCGATCGCGCG)2 in the B-form with two putative intercalation sites at the 5′- CGA-3′ step on both strands. Consequently we are able to look at the structure of a 1:1 complex and exclude crystal packing effects normally encountered in most of the X-ray crystallographic studies conducted so far. We performed different 10 to 20 ns long molecular dynamics simulations of the uncomplexed DNA structure, the DNA-daunomycin complex and a 1:2 complex of DNA-daunomycin where the two intercalator molecules are stacked into the two opposing 5′-CGA-3′ steps. Thereby—in contrast to X-ray structures—a comparison of a complex of only one with a complex of two intercalators per doublehelix is possible. The chromophore of daunomycin is intercalated between the 5′-CG-3′ bases while the daunosamine sugar moiety is placed in the minor groove. We observe a flexibility of the dihedral angle at the glycosidic bond, leading to three different positions of the ammonium group responsible for important contacts in the minor groove. Furthermore a distinct pattern of BI and BII around the intercalation site is induced and stabilized. This indicates a transfer of changes in the DNA geometry caused by intercalation to the DNA backbone.

Z-DNA's Conformer Substates Revealed by FT-IR Difference Spectroscopy of Nonoriented Left-Handed Double Helical Poly(dG-dC)

Abstract Nonoriented hydrated films of double helical poly(dG-dC) in the Z-form were studied by Fourier transform infrared (FT-IR) spectroscopy either as equilibrated slow-cooled samples between 290 and 220 K or, after quenching into the glassy state, as nonequilibrated film isothermally at 200, 220, and 240 K. IR spectral changes on isothermal relaxation at 200 and 220 K toward equilibrium, caused by interconversion of two conformer substates (CS) called Z1 and Z2, are revealed by IR difference spectra. Pronounced spectral changes on Z1-to-Z2 interconversion occur between ≈750–1250 cm−1 and these are attributed to structural changes of the phosphodiester-sugar backbone caused by changes of torsion angles, and to decreasing hydrogen-bonding of the ionic phosphate group with water molecules. These spectral changes on Z1-to-Z2 transition can be related to structural differences between ZI and ZII CS observed in single crystals. ZI/ZII CS occurs only at (dGpdC) base steps, and similar behavior is assumed for Z1/Z2. The Z1/Z2 population ratio was determined via curve resolution of marker bands for Z1 and Z2 centered at 785 and 779 cm−1. This ratio is 0.64 at 290 K, corresponding to 39% of the phosphates of the (dGpdC) base steps in Z1 and 61% in Z2, and it increases to 1.24 on cooling to 220 K. For the Z2⇔Z1 equilibrium, an enthalpy change of −4.9 ± 0.2 kJ mol(dGpdC)−1 is obtained from the temperature dependence of the equilibrium constant. Z1 interconverts into Z2 at isothermal relaxation at 200 and 220 K, whereas on slow cooling from ambient temperature, Z2 interconverts into Z1. This unexpected reversal of CS interconversion is attributed to slow restructuring of hydration shells of the CS on quenching, in the same manner reported by Pichler et al. for the BI and BII CS of B-DNA (J. Phys. Chem. B 106, 3263–3274 (2002)). IR difference curves demonstrate two time scales on isothermal relaxation of Z1→Z2 interconversion, a fast one for structural relaxation of the sugar-phosphate backbone, and a slow one for relaxation of the hydration shells. This slowing down of restructuring of CS hydration shells at ≈220–240 K could be the cause for the suppression of biological functions at low temperatures.

Towards an understanding of DNA recognition by the methyl-CpG binding domain 1.

Abstract CpG methylation determines a variety of biological functions of DNA. The methylation signal is interpreted by proteins containing a methyl-CpG binding domain (MBDs). Based on the NMR structure of MBD1 complexed with methylated DNA we analysed the recognition mode by means of molecular dynamics simulations. As the protein is monomeric and recognizes a symmetrically methylated CpG step, the recognition mode is an asymmetric one. We find that the two methyl groups do not contribute equally to the binding energy. One methyl group is associated with the major part of the binding energy and the other one nearly does not contribute at all. The contribution of the two cytosine methyl groups to binding energy is calculated to be −3.6 kcal/mol. This implies a contribution of greater than two orders of magnitude to the binding constant. The conserved amino acid Asp32 is known to be essential for DNA binding by MBD1, but so far no direct contact with DNA has been observed. We detected a direct DNA base contact to Asp32. This could be the main reason for the importance of this amino acid. MBD contacts DNA exclusively in the major groove, the minor groove is reserved for histone contacts. We found a deformation of the minor groove shape due to complexation by MBD1, which indicates an information transfer between the major and the minor groove.

PvuII-endonuclease induces structural alterations at the scissile phosphate group of its cognate DNA.

We investigated the PvuII endonuclease with its cognate DNA by means of molecular dynamics simulations. Comparing the complexed DNA with a reference simulation of free DNA, we saw structural changes at the scissile phosphodiester bond. At this GpC step, the enzyme induces the highest twist and axial rise, inclination is increased and the minor groove widened. The distance between the scissile phosphate group and the phosphate group of the following thymine base is shortened significantly, indicating a substrate-assisted catalysis. A feasible reason for this vicinity is the catalytically important amino acid residue lysine 70, which bridges the free oxygen atoms of the successive phosphate groups. Due to this geometry, a compact reaction pocket is formed where a water molecule can be held, thus bringing the reaction partners for hydrolysis into contact. The O1-P-O2 angle of the scissile nucleotide is decreased, probably due to a complexation of the negative oxygen atoms through protein and solvent contacts.

The Conformer Substates of Nonoriented B-type DNA in Double Helical Poly(dG-dC)

Abstract A nonoriented hydrated film of poly(dG-dC) with ≈20 water molecules per nucleotide (called B by Loprete and Hartman (Biochem. 32, 4077–4082 (1993)) was studied by Fourier transform infrared (FT-IR) spectroscopy either as equilibrated sample between 290 and 270 K or, after quenching into the glassy state, as nonequilibrated film isothermally at 200 and 220 K. IR spectral changes on isothermal relaxation at 200 and 220 K, caused by interconversion of two conformer substates, are revealed by difference spectra. Comparison with difference curves obtained in the same manner from two classical B-DNA forms, namely the d(CGCGAATTCGCG)2 dodecamer and polymeric NaDNA from salmon testes, revealed that the spectral changes on BIto-BII interconversion in the classical B-DNA forms are very similar to those in the B-form, and that the spectroscopic differences between the BI and BII features from classical B-DNA and those from the modified B-form are minor. Nonexponential kinetics of the BI→BII transition in the B-form of poly(dG-dC) at 200 K showed that the structural relaxation time is about three times of that in the classical B-DNA forms (≈30 versus ≈10 min at 200 K). The unexpected reversal of conformer substates interconversion (that is BII→BI transition on cooling from 290 K and BI→BII transition on isothermal relaxation at 200 K) observed for classical B-DNA occurs also in the modified B-form. We therefore conclude that restructuring of hydration shells rules the low-temperature dynamics of the B-form via its two conformer substates in the same manner reported for classical B-DNA by Pichler et al. (J. Phys. Chem. B 106, 3263–3274 (2002)).