γ-Ray footprinting and fluorescence polarization anisotropy of a 30-mer synthetic DNA fragment with one 2′-deoxy-7-hydro-8- oxoguanosine lesion

Abstract

The influence of the oxidative lesion 2'-deoxy-7-hydro-8-oxoguanosine (8-oxodG) on some conformational properties of DNA has been studied. Four 30-mer duplexes of the form [5'-GATCCTCTAGAGTC[G(*) or G]ACCTGCAGGCATGCA-3']:[3'-CTAGGAGATCTCAG[C or A]TGGACGTCCGTACGT-5'], in which G(*) is the 8-oxodG lesion, were synthesized in order to compare the effect of the GA mismatch and of the damaged G(*)C and G(*)A forms with the normal GC. Spectroscopic measurements performed by means of UV denaturation and circular dichroism experiments do not show gross changes of stability and overall structure in the damaged and mismatched samples. The control DNA and the samples containing GA mismatch show very similar gamma-rays cutting patterns, indicating that the introduction of the GA mismatch does not perturb the phosphate backbone geometry. In the samples containing the 8-oxodG there are some variations of the cleavage pattern near G(*) which are extended for almost one helical turn. Some differences are observed between G(*)C and G(*)A duplexes. In particular, in the G(*)C sample the reduced accessibility to OH radicals at the G15 site, observed in the control, spreads on the intrastrand adjacent bases and in the G(*)A sample a shift of the minimum is observed. The hydrodynamic radius R(h) derived by fluorescence polarization anisotropy decay exhibits a constant value of 11.4 +/- 0.2 A between 5 and 40 degrees C, in all the samples. The torsional constant alpha of each oligomer decreases when the temperature is raised and the alpha values of the damaged samples are higher than those of the normal ones.

Can reliable torsion elastic constants be determined from FPA data on 24 and 27 base-pair DNAs?

“…Those were combined with ten additional values reported previously for 27 bp oligomers [2] to create a data set containing 53 torsion elastic constants. In earlier studies, the same group of Pedone, Barone, Mazzei and coworkers also reported torsion elastic constants for other 27 bp DNA, RNA, and DNA-RNA hybrid duplexes and also for a 30 bp DNA containing a lesion [3][4][5][6]. Prior investigations of duplexes containing V36 bp by other groups were unable to obtain reliable estimates of the torsion elastic constants, and no values were reported [7][8][9][10].…”

“…The objectives of the present study are (1) to show that two 24 bp DNAs with a~5-fold ratio of torsion elastic constants and a 10% difference in r 0 can exhibit anisotropy decays that are practically indistinguishable for all times, tN1 ns; (2) to show that a and r 0 cannot be both be determined from FPA data for a 24 or 27 bp DNA using instruments like that of Pedone et al or that in our laboratory; (3) to note that for much longer DNAs, where a and r 0 can be simultaneously determined, the best-fit r 0 does not always exhibit the same value, 0.360, but instead varies over a range from 0.31 to 0.38, and is sensitive to Poisson noise and other variations in the experimental data; (4) to show that a rather small error in the assumed fixed value of r 0 can cause an extremely large error in the best-fit value of a; (5) to note that the values of R H and L reported by Pedone et al [2] for ten 27 bp duplexes differ from their expected values, and also among each other, by more than would be expected; and (6) to show that the 4.4-fold span of a-values reported by Pedone et al can be obtained by fitting FPA data that are simulated using a single value of a and modestly different r 0 values within the previously observed range of such values. The likelihood of validity of the 53 values reported by Pedone et al [1] is discussed in detail.…”

“…That theory, which was employed in one way or another in references [1][2][3][4][5][6][7][8][9][10][11][12][13][14][15][16], is presented in Appendix A, where its various parameters are also defined.…”

“…In references [1][2][3][4][5][6][7][8][9][10][11][12][13][14], the hydrodynamic properties and in some cases also the torsion elastic constant were assessed by measuring the temporal decay of the fluorescence polarization anisotropy (FPA) of intercalated ethidium in short DNA duplexes. In our laboratory these measurements were performed by time-correlated singlephoton counting using an apparatus with an instrumental width (full width at half maximum (FWHM)) ] 80 ps, which defines its effective time-resolution.…”

Modelling radiation-induced damage in the lac operator - lac repressor complex. DNA damage: 8-oxoguanine

“…The reduced protein binding anity can be due to DNA structural or¯exibility changes. Until now no global structural changes but only some dierences in thermal and thermodynamic properties [8,9] and subtle changes to DNA dynamics were evidenced for short oligomers containing one oxoG [10].…”