Topoisomerase 1 (Top1) enzymes regulate DNA superhelicity by forming covalent cleavage complexes that undergo controlled rotation. Substitution of nucleoside analogs at the +1 position of the DNA duplex relative to the Top1 cleavage site inhibits DNA religation. The reduced efficiency for Top1-mediated religation contributes to the anticancer activity of widely used anticancer drugs including fluoropyrimidines and gemcitabine. In the present study, we report that mismatched base pairs at the +1 position destabilize the duplex DNA components for a model Top1 cleavage complex formation even though one duplex component does not directly include a mismatched base pair. Molecular dynamics simulations reveal G-dU and G-FdU mismatched base pairs, but not a G-T mismatched base pair, increase flexibility at the Top1 cleavage site, and affect coupling between the regions required for the religation reaction to occur. These results demonstrate that substitution of dT analogs into the +1 position of the non-scissile strand alters the stability and flexibility of DNA contributing to the reduced efficiency for Top1-mediated DNA religation. These effects are inherent in the DNA duplex and do not require formation of the Top1:DNA complex. These results provide a biophysical rationale for the inhibition of Top1-mediated DNA religation by nucleotide analog substitution.
DNA topoisomerasesregulate the topological state of DNA as required to relieve superhelical density for important biological processes such as replication and transcription [
Top1 is the sole target for the camptothecin (CPT) class of anticancer drugs. CPT forms a stable ternary complex upon binding to the Top1:DNA covalent cleavage complex. Stabilization of cleavage complexes by CPT converts Top1 into a cellular poison since collision of advancing replication forks with trapped Top1 cleavage complexes results in DNA double-strand breaks. Thus, CPT not only inhibits Top1 activity, but also converts the enzymatic activity into DNA damage that is potentially lethal to the cell. Over the last decade, it has been shown that a variety of nonnative nucleotide substitutions that may result from oxidative damage to DNA (e.g., 8-oxo-dG) or covalent modification of DNA nucleobases (e.g., benzpyrene adducts) also cause trapping of Top1 cleavage complexes and result in DNA DSB formation [
The structural basis for trapping of Top1 cleavage complexes by damaged nucleobases or misincorporation of nucleotide analogs into the nonscissile strand of DNA remains an area of investigation. Although the DNA sequence used in most model studies of Top1:DNA interactions contains several A-tracts [
Model system used for evaluating the effects of mismatched base pairs on the stability of the intermediate required for Top1-mediated DNA religation. Top: depiction of the equilibrium between the 10 mer single-stranded DNA and the 39 mer DNA hairpin that constitutes a model system for Top1-mediated DNA religation. A G-X base pair is at the +1 position relative to the Top1 cleavage site. Bottom (a–d): depiction of the steps of Top1 binding, strand dissociation, strand reassociation, and religation.
A model Top1 cleavage site (Figure
Absorbance versus temperature profiles of each oligonucleotide in buffer were measured at 260 nm using a thermoelectrically controlled Aviv model 14DS UV-vis spectrophotometer (Lakewood, NJ). The temperature was scanned from 20°C to 95°C for the 39 mer DNA hairpins and from 1–100°C for the model Top1 cleavage sites at a heating rate of 0.6°C/min. DNA concentrations were 1.5–2.0
The simulation of the four hairpins is performed using NAMD [
The effects of mismatched base pairs at the +1 position on the stability of the model Top1 cleavage site were investigated using UV hyperchromicity measurements. Initial studies focused on the stabilities of the four 39 mer DNA hairpins (Figure
Stability of the 39 mer DNA hairpin corresponding to a model Top1 cleavage site. A single coordinated transition is observed corresponding to the thermal melting transition for the double-stranded region of the hairpin. As expected, the melting temperature is dependent upon the presence of a mismatched base pair at the terminus of the duplex region. The native sequence is most stable while C → T, C → dU, and C → FdU decrease the stability of the duplex region by 4.4°C, 3.0°C, and 3.5°C, respectively.
We next investigated the stability of the model Top1 cleavage complex using UV hyperchromicity measurements. The four 39 mer DNA hairpins consisting of 13 base pairs with a 10 mer single-stranded overhang were annealed to the 10 mer ssDNA complementary to the overhang region (Figure
Thermal stability of the complex of the 10 mer ssDNA and the 39 mer DNA hairpin constituting a model Top1 religation complex. (a) Melting transition in 10 mM sodium phosphate buffer pH 7; (b) melting transition in the same buffer as A, but with 200 mM NaCl. A biphasic melting profile is observed in both instances. The lower melting temperature corresponds to the dissociation of the ssDNA from the 39 mer DNA hairpin, while the higher melting transition corresponds to the melting temperature for the duplex region of the hairpin. As was observed for the DNA hairpin alone (Figure
Comparing molecular dynamics simulations for the four different 49 mer DNA hairpins (39 mer DNA hairpin extended without a nick in the phosphodiester backbone) demonstrates that all the mismatches indeed do produce profound effects on the initial 10 base pairs that form the recognition sequence and its complement. Due to the observed effects on the thermal stability and the novel influence of different mismatches occurring outside the recognition sequence, three different atomic measures of structural fluctuations within these first 10 base pairs are used to quantify this influence. First, atomic root-mean-square fluctuations (RMSFs) are calculated for all heavy atoms and averaged on a per-base level. This quantifies the extent to which each atom fluctuates about its equilibrium position and averages these fluctuations at the base-level for comparisons to determine how the different mismatches affect both the overall atomic fluctuations of the first 10 base pairs and the pattern of fluctuations. Second, correlated fluctuations are calculated. These determine the extent to which atomic fluctuations, regardless of magnitude, are correlated or anticorrelated and are averaged at the base level for comparison among the four different simulation types. This measure, referred to as the covariance matrix, determines how the different mismatches affect the coupling within the first 10 base pairs. This method has been used by multiple research groups to analyze communication within proteins [
The first noticeable effect of mismatch formation is on the RMSFs of the G-dU and G-FdU hairpins; the average RMSF over the first 10 base pairs increases by 13.5% and 12.0% for the G-dU and G-FdU hairpins, respectively, relative to the average RMSF of the matched hairpin (Table
DNA RMSFs (Å)*.
Matched | 2.136 |
G-T | 2.120 |
G-dU | 2.423 |
G-FdU | 2.391 |
*The root-mean-square fluctuations in Å for the first 10 base pairs of each sequence are averaged.
(a) Per-base-averaged RMSFs. The atomic root-mean-square fluctuations for the first ten bases for each of the DNA sequences are depicted: matched DNA as blue +s, T mismatched as red °s, dU mismatched as magenta ×s, and FdU mismatched as black squares. (b) Per-base-averaged RMSFs relative to matched DNA as (a), but with the matched DNA rmsf values subtracted.
The covariance matrices (Figure
(a) Matched covariance matrix. The per-base-averaged covariance matrix plotted as a color map with red indicating high correlations of fluctuations blue indicating high anticorrelation of fluctuations, and yellow indicat uncorrelated. (b) Mismatched-matched difference covariance matrix. The matched covariance matrix (a) subtracted from the mismatched covariance matrix (not shown). (c) FdU-matched difference covariance matrix. The matched covariance matrix (a) subtracted from the FdU-mismatch covariance matrix (not shown). (d) Uracil-matched difference covariance matrix. The matched covariance matrix (a) subtracted from the uracil-mismatch covariance matrix (not shown).
Both the RMSFs and covariances demonstrate that there are delocalized changes in atomic covariances and their correlations in the recognition sequences despite the mismatch occurring outside this sequence; clustering analysis sheds light on the population shifts and conformational changes that may give rise to these variations, and that may perturb the recognition and binding by Top1.
Clustering analysis on the first 10 base pairs shows that there are four different conformations that are accessible to each of the four DNA sequences. The difference lies in their populations (Table
Cluster populations (%).
Cluster no./Sequence | Matched | G-T | G-dU | G-FdU |
---|---|---|---|---|
1 | 29.0% | 28.5% | 14.2% | 32.9% |
2 | 37.4% | 30.1% | 32.7% | 20.2% |
3 | 24.8% | 22.6% | 27.8% | 20.4% |
4 | 8.8% | 18.9% | 25.3% | 26.4% |
The actual structural rearrangements that occur during these conformational changes are actually quite modest (Figure
(a) Overall conformations adopted in the four clusters. The first ten base pairs of the centroids of each of the four clusters are shown in VMD’s NewRibbon format. Cluster 1 (see Table
One issue that can also be addressed is that of the classification of the different DNA sequences, which ones are more similar, and which are more different. With three different measures, the simplest approach to this problem is to cluster the four sequences based on each of these three measures (Table
Clustering of simulations via different measures.
Measure/Sequence | Matched | G-T | G-dU | G-FdU |
---|---|---|---|---|
RMSF | 1 | 1 | 2 | 2 |
Covariance matrix | 1 | 1 | 2 | 3 |
Cluster population | 1 | 2 | 3 | 2 |
Cluster number for each simulation for each measure.
DNA mismatched base pairs occur at high levels in cells both as a consequence of base damage (e.g., cytidine deamination) [
A G-dU (or G-FdU) base pair in wobble geometry has two hydrogen bonds as does an A-T Watson-Crick base pair and thus is a relatively conservative substitution although at elevated pH an ionized G-FdU base pair may form [
Molecular dynamics simulations demonstrate that the mismatched base pairs increase the flexibility of the duplex. The extent of this increase in flexibility is dependent upon the type of mismatch with G-dU and G-FdU mismatches displaying the greatest increased flexibility. These calculations reveal that the mismatched base pair causes increased atomic fluctuations up to 10 base pairs removed from the site of the mismatch. This increased flexibility makes it less likely that the scissile strand will adopt the correct conformation required for the religation reaction. Thus, the thermodynamic measurements obtained from the UV hyperchromicity data demonstrate that formation of the complex required for religation is disfavored by all DNA mismatched base pairs at the +1 site of the religation complex while the molecular dynamics simulations reveal that the G-dU and G-FdU mismatched pairs are especially potent at increasing conformational flexibility and decreasing the likelihood religation will occur. Overall, our results provide new insights into the structural and dynamic process of Top1-mediated DNA religation and the influence of mismatched base pairs, particularly G-dU and G-FdU mismatched base pairs, at disfavoring this process.
This work was supported by Grants nos. NIH CA-102532 (W. H. Gmeiner) and NIH P30 CA-12197.