The major structural aberrations of DNA induced by a cis-diammineplatinum (II) 1,2-d(GG) intrastrand cross-link (CPT) have been known for decades. To gain deeper insights into the structural dynamics ...of the sequence-dependent DNA distortions adjacent to the CPT adduct, we employed molecular modeling and molecular dynamics (MD) simulations. The structural dynamics of native (N-DNA) and cisPt 1,2-d(GG) crosslinked (CPT-DNA) in the form of symmetric 36 nt d(G
2
T
15
G*G*T
15
G
2
)●C
2
A
15
CCA
15
C
2
) oligonucleotide duplexes is compared. The selected sequence context enabled tracking of the origin of the DNA axis curvature at the YpR flexible points (N-DNA), the enhancement of axis bending, and further distortions due to steric/electrostatic perturbations arising from the CPT-crosslink. In addition to the known structural distortions of CPT-DNA: helix bend towards the major groove; local helix unwinding; high roll angle between cross-linked guanine bases; and adoption of A-form DNA on the 5′-side of the CPT-crosslink (TpG junction); our results show the existence of a singular irreversible and reproducible conformational rearrangement, not previously observed, resulting in two stable CPT-DNA1 and CPT-DNA2 conformers. The CPT-DNA2 conformation presents an enhanced DNA axis bend and a wider and shallower minor grove with increased solvent accessibility within the modified site. It is concluded that the polymorphous (unstable) DNA environment near the
cis
Pt 1,2-d(GG) unit in synergy with specific dynamic events, such as prolonged minor groove retention of particular Na
+
ions and water redistribution within the d(TG*G*T) site, together with the formation of extra and more stable H-bonds between Pt(NH
3
)
2
amines and neighboring nucleotides, are cooperatively responsible for the initiation of the conformational rearrangement leading to the CPT-DNA2 conformer, which, surprisingly, closely resembles the HMGB1-bound CPT-DNA structure.
Graphical abstract
Superimposed averaged structures of normal (N-DNA,
green
) and cisplatin intrastrand cross-linked (CPT-DNA,
orange
). Global DNA axes: N-DNA (
blue beads
); CPT-DNA (
red beads
); PT (
yellow sphere
); crosslinked dGs viewed from the minor groove (
bold
)
The UvrA protein initiates the DNA damage recognition process by the bacterial nucleotide excision repair (NER) system. Recently, crystallographic structures of holo-UvrA(2) dimers from two different ...microorganisms have been released (Protein Data Bank entries 2r6f , 2vf7 , and 2vf8 ). However, the details of the DNA binding by UvrA(2) and other peculiarities involved in the damage recognition process remain unknown. We have undertaken a molecular modeling approach to appraise the possible modes of DNA-UvrA(2) interaction using molecular docking and short-scale guided molecular dynamics continuum field, constrained, and/or unrestricted simulated annealing (SA), taking into account the three-dimensional location of a series of mutation-identified UvrA residues implicated in DNA binding. The molecular docking was based on the assumptions that the UvrA(2) dimer is preformed prior to DNA binding and that no major protein conformational rearrangements, except moderate domain reorientations, are required for binding of undamaged DNA. As a first approximation, DNA was treated as a rigid ligand. From the electrostatic relief of the ventral surface of UvrA(2), we initially identified three, noncollinear DNA binding paths. Each of the three resulting nucleoprotein complexes (C1, C2, and C3) was analyzed separately, including calculation of binding energies, the number and type of interaction residues (including mutated ones), and the predominant mode of translational and rotational motion of specific protein domains after SA to ensure improved DNA binding. The UvrA(2) dimer can accommodate DNA in all three orientations, albeit with different binding strengths. One of the UvrA(2)-DNA complexes (C1) fulfilled most of the requirements (high interaction energy, proximity of DNA to mutated residues, etc.) expected for a natural, high-affinity DNA binding site. This nucleoprotein presents a structural organization that is designed to clamp and bend double-stranded DNA. We examined the binding site in more detail by docking DNAs of significantly different (AT- vs CG-enriched) sequences and by submitting the complexes to DNA-unrestricted SA. It was found that in a manner independent of the DNA sequence and applied MD protocols, UvrA(2) favors binding of a bent and unwound undamaged DNA, with a kink positioned in the proximity of the Zn3 hairpins, anticollinearly aligned at the bottom of the ventral protein surface. It is further hypothesized that the Zn3 modules play an essential role in the damage recognition process and that the apparent existence of a family of DNA binding sites might be biologically relevant. Our data should prove to be useful in rational (structure-based) mutation studies.
Peptide nucleic acids (PNAs) efficiently hybridize with DNA and are promoted as versatile gene-targeting analytical tools and pharmaceuticals. However, PNAs have never been exploited as ...radiopharmaceuticals, and radiation-induced physicochemical modifications of PNA:DNA heteroduplexes have not been studied. Drug- and radiation-induced creation of covalent cross-links in DNA obstruct crucial cell survival processes such as transcription and replication and are thus considered genotoxic events with a high impact in anticancer therapies. Here we report that γ-irradiation of complementary PNA:DNA heteroduplexes, wherein the PNA contains l-lysine, free amino, or N-methylmorpholinium N- and C-capping groups, results in the formation of irreversible interstrand cross-links (ICL). The number of detected ICL corresponds to the number of available amino functional groups on the PNA. The effect of DNA sequence on the formation of ICL was studied by modifying the terminal nucleotides of the DNA oligonucleotide to create deletions and overhangs. The involvement of abasic sites (ABS) on the DNA strand in the cross-linking reaction was confirmed by independent experiments with synthetic ABS-containing oligonucleotides. Molecular modeling and molecular dynamics (MD) simulations were applied to elucidate the conformation of the N- and C-capping groups of the PNA oligomer and their interactions with the proximal terminus of the DNA. Good agreement between experimental and modeling results was achieved. Modeling indicated that the presence of positively charged capping groups on the PNA increases the conformational flexibility of the PNA:DNA terminal base pairs and often leads to their melting. This disordered orientation of the duplex ends provides conditions for multiple encounters of the short (amino) and bulky (Lys) side chains with nucleobases and the DNA backbone up to the third base pair along the duplex stem. Dangling duplex ends offer favorable conditions for increased accessibility of the radiation-induced free radicals to terminal nucleotides and their damage. It is suggested that the ICL are produced by initial formation of Schiff base adducts between the PNA amino functions and the opposed DNA oxidation-damaged bases or abasic 2′-deoxyribose-derived aldehydic groups. The subsequent reduction by solvated electrons (e− aq) or other radiation-produced reducing species results in irreversible covalent interstrand cross-links. The simultaneous involvement of oxidizing, •OH, and reducing, e− aq, radicals presents a case in which multiple ionization events along a γ-particle path lead to DNA injuries that also encompass ICL as part of the multiply damaged sites (MDS). The obtained results may find applications in the development of a new generation of gene-targeted radiosensitizers based on PNA vectors.
Solvated electrons (
) are produced during water radiolysis and can interact with biological substrates, including DNA. To augment DNA damage, radiosensitizers such as bromo-deoxyuridine (BUdR), ...often referred to as an “electron affinic radiosensitizer”, are incorporated in place of isosteric thymidine. However, little is known about the primary interactions of
with DNA. In the present study we addressed this problem by applying molecular modeling and molecular dynamics (MD) simulations to a system of normal (BUdR·A)-DNA and a hydrated electron, where the excess electron was modeled as a localized
(H
2
O)
6
anionic cluster. Our goals were to evaluate the suitability of the MD simulations for this application; to characterize the motion of
around DNA (e.g., diffusion coefficients); to identify and describe configurational states of close
localization to DNA; and to evaluate the structural dynamics of DNA in the presence of
. The results indicate that
has distinct space-preferences for forming close contacts with DNA and is more likely to interact directly with nucleotides other than BUdR. Several classes of DNA -
contact sites, all within the major groove, were distinguished depending on the structure of the intermediate water layer H-bonding pattern (or its absence, i.e., a direct H-bonding of
with DNA bases). Large-scale structural perturbations were identified during and after the
approached the DNA from the major groove side, coupled with deeper penetration of sodium counterions in the minor groove.
Figure
A rare configuration showing direct interaction between the solvated electron and DNA, where
(yellow) and N7(A16) are H-bonded. The close
approach from the major groove side invokes deep Na
+
(magenta) penetration into the minor DNA groove (Fig.
7
a).
The interaction of solvated electrons
with DNA results in various types of DNA lesions. The in vitro and in vivo sensitisation of DNA to
-induced damage is achieved by incorporation of the ...electron-affinity radiosensitiser bromodeoxyuridine (BUdR) in place of thymidine. However, in DNA duplexes containing single-stranded regions (bulged BUdR-DNA), the type of lesion is different and the efficiency of damage is enhanced. In particular, DNA interstrand crosslinks (ICL) form at high efficiency in bulged DNA but are not detectable in completely duplex DNA. Knowledge about the processes and interactions leading to these differences is obscure. Previously, we addressed the problem by applying molecular modelling and molecular dynamics (MD) simulations to a system of normal (BUdR·A)-DNA and a hydrated electron, where the excess electron was modelled as a localised e¯(H
2
O)
6
anionic cluster. The goal of the present study was to apply the same MD simulation to a wobble
system, containing a pyrimidine–pyrimidine mismatched base pair, BUdR·T. The results show an overall dynamic pattern similar to that of the
motion around normal DNA. However, the number of configuration states when
was particularly close to DNA is different. Moreover, in the (BUdR·T)-wobble DNA system, the electron frequently approaches the brominated strand, including BUdR, which was not observed with the normal (BUdR·A)-DNA. The structure and exchange of water at the sites of
immobilisation near DNA were also characterised. The structural dynamics of the wobble DNA is prone to more extensive perturbations, including frequent formation of cross-strand (cs) interatomic contacts. The structural deviations correlated with
approaching DNA from the major groove side, with sodium ions trapped deep in the minor groove. Altogether, the obtained results confirm and/or throw light on dynamic-structure determinants possibly responsible for the enhanced radiation damage of wobble DNA.
Figure
The structure of the tightly bound single water-layer between the DNA and the electron (Site-8, five H
2
O molecules,
bold capped sticks
); the rest of the “second” shell waters (
lines
, in atom type colour) surround the ¯(H
2
O)
6
cluster (
yellow
,
space fill
).
Orange dashed lines
H-bonds; only one of the five molecules from the single H
2
O layer mediates a single-step H-bond bridge with N7(A8); the other four present a network of two(three)-step H-bond bridges between DNA/
partner atoms
Epipodophyllotoxin derivatives, such as etoposide (VP-16), constitute an important class of anticancer agents, the major cytotoxic
effects of which are associated with trapping of the topoisomerase ...II/DNA cleavable complex and formation of protein-DNA cross-links
and nicked DNA. VP-16, however, can be metabolized to several highly reactive products, including an ortho -quinone (VPQ). The inhibitory activity of VPQ against purified human topoisomerase II processing of supercoiled DNA was studied
and compared with that of the parent compound, VP-16. Our results show that VPQ is a powerful inhibitor of topoisomerase II,
which prevents DNA strand passage in the presence of ATP. As with VP-16, trapping of the cleavable complex is highly reversible
upon removal of divalent ions, which indicating that VPQ alters the cleavage-reunion equilibrium of topoisomerase II and DNA
mainly by noncovalent interactions, as does the parent compound. However, we observed several differences between the effects
induced by VP-16 and VPQ, including a strong inhibition of the second DNA strand religation, which implies the involvement
of additional (asymmetric) mode(s) of interactions of the VPQ, possibly by interference with ATP binding by the homodimeric
enzyme, and/or involving covalent interactions. Reduced or oxidized glutathione prevented trapping of the topoisomerase/DNA
cleavable complex by VPQ, but not by VP-16, probably by forming covalent adducts with the former.
The 3-D quantitative structure-activity relationships/comparative molecular field analysis (QSAR/CoMFA) paradigm, which considers the primary importance of the molecular fields in biological ...recognition, is now widely used to analyze and predict receptor-binding properties of various ligands. CoMFA was applied to build 3-D QSAR models of substituted estradiol-receptor interactions, employing 3-D molecular databases of more than 40 molecules. Ligands included the 17 alpha-ethynyl- and isomeric 17 alpha (20E/Z)-(iodovinyl)estradiols and their 7 alpha-, 11 beta-, and 12 beta-methyl (-methoxy) and -ethyl (-ethoxy) derivatives as well as selected 2- and 4-halogenated analogs. The influence of different CoMFA descriptors was studied in order to achieve the highest possible cross-validated r2, as derived from partial least-squares calculations. Special emphasis was put on the analysis of the nature of H-bonding (donor/acceptor) interactions. The model with the best predictive performance (r2 = 0.895) was used to visualize steric and electrostatic features of the QSAR (standard deviation*coefficient contour maps) and to predict receptor-binding affinities (RBA) of substituted estradiols other than those included in the original database. Twenty-seven test molecules were selected, including five which had previously been reported by other investigators. For the latter, a very good correlation with literature RBA values was obtained, which together with the high cross-validated r2 provides evidence for the high predictive capacity of the model. Among the unknown structures, the model suggests several new substitutions to derive at reasonable affinity ligands for the estrogen receptor.
Photo-excited Zn-phthalocyanine (ZnPc) interactions with four (hydro)peroxides (ROOR
′) were studied by T–T absorption decay and fluorescence quenching. Triplet state interactions are weak (log
k
...q∼4) and independent from the quencher used, while singlet quenching is variable (log
k
q 4.5–9.4). EPR and spin-trapping show that at low ZnPc concentrations the primary decomposition products are alkoxyl radicals. At high ZnPc concentrations, photosensitization of ROOH gives rise to peroxyl radicals. The (hydro)peroxide reducing potentials were estimated following AM1 MO-computations. Our experimental data and theoretical predictions (Marcus–Savéant theory) are consistent with a charge transfer mechanism to account for the alkoxyl radical formation.
