A general approach to the quantitative study of the sequence specificity of DNA interstrand crosslinking agents in synthetic duplex DNA fragments is described. In the first step, a DNA fragment previously treated with an interstrand crosslinking agent is subjected to denaturing PAGE. Not only does this distinguish crosslinked from native or monoadducted DNA, it is shown herein that isomeric crosslinked DNAs differing in position of the crosslink can in some cases be separated. In the second stage, the now fractionated crosslinked DNAs isolated from denaturing PAGE are subjected to fragmentation using iron(II)/EDTA. For those fractions which are structurally homogeneous, analysis of the resulting fragment distribution has previously been shown to reveal the crosslink position at nucleotide resolution. It is shown herein that in fractions which are structurally heterogeneous due to differences in position of crosslink, this analysis quantifies the relative extent of crosslinking at distinct sites. Using this method it is shown that reductively activated mitomycin C crosslinks the duplex sequences 5'-GCGC and 5'-TCGA with 3 +/- 1:1 relative efficiency.

AB STRACTThe fluorescence polarization anisotropies of ethidium intercalated in duplex DNAs containing 12 to 60 bp were measured and analyzed using a theory of deformable rods to obtain their hydrodynamic radii (RH). The apparent RH 5 10.0 0.2 A for 12 and 24 bp, but increases to 1 1 .5-12.0 A between 40 and 60 bp. This variation in RH is attributed to the presence of slowly relaxing bends in the DNA.Consequently, the static equilibrium persistence length must be much smaller than the dynamic persistence length ('d) defined by the dynamic (instantaneous) bending rigidity. We estimate that d A. RH of a 48 bp duplex DNA is largely independent of NaCl concentration, which suggests that interionic forces do not much affect the rotational friction. However, RH depends significantly on the concentrations of other ions, especially Mg2 and Mn2, which are believedto affect the hydration of DNA.

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