Oxidative guanine lesions were analyzed, at the nucleotide level, within DNA exposed to nanosecond ultraviolet (266 nm) laser pulses of variable intensity (0.002-0.1 J/cm 2). Experiments were carried out, at room temperature, in TE buffer (20 mM Tris-HCl, pH 7.5; 1 mM EDTA) containing 35 mM NaCl, on 59-end radioactively labeled double-stranded and singlestranded oligomer DNA at a size of 33-37 nucleobases. Lesions were analyzed on polyacrylamide gel electrophoresis by taking advantage of the specific removal of 8-oxodG from DNA by the formamidopyrimidine DNA glycosylase (Fpg protein) and of the differential sensitivity of 8-oxodG and oxazolone to piperidine. The quantum yields of lesions at individual sites, determined from the normalized intensities of bands, were plotted against the irradiation energy levels. Simplified model fitting of the experimental data enabled to evaluate the spectroscopic parameters characterizing excitation and photoionization processes. Results show that the distribution of guanine residues, excited to the lowest triplet state or photoionized, is heterogeneous and depends on the primary and secondary DNA structure. These findings are generalized in terms of excitation energy and charge-migration mediated biphotonic ionization. On the basis of the changes in the yield of the guanyl radical resulting from local helical perturbations in the DNA p-stack, it can be assessed that the distance range of migration is ,6-8 bp.

Despite its small size (27.6 kDa), the group I intronencoded I-SceI endonuclease initiates intron homing by recognizing and specifically cleaving a large intronless DNA sequence. Here, we used gel shift assays and footprinting experiments to analyze the interaction between I-SceI and its target. I-SceI was found to bind to its substrate in monomeric form. Footprinting using DNase I, hydroxyl radical, phenanthroline copper complexes, UV/DH-MePyPs photosensitizer, and base-modifying reagents revealed the asymmetric nature of the interaction and provided a first glimpse into the architecture of the complex. The protein interacts in the minor and major grooves and distorts DNA at three distinct sites: one at the intron insertion site and the other two, respectively, downstream (؊8, ؊9) and upstream (؉9, ؉10) from this site. The protein appears to stabilize the DNA curved around it by bridging the minor groove on one face of the helix. The scissile phosphates would lie on the outside of the bend, facing in the same direction relative to the DNA helical axis, as expected for an endonuclease that generates 3 overhangs. An internally consistent model is proposed in which the protein would take advantage of the concerted flexibility of the DNA sequence to induce a synergistic binding/kinking process, resulting in the correct positioning of the enzyme active site.I-SceI is a homing endonuclease encoded by the mobile group I intron of the large rRNA gene of Saccharomyces cerevisiae (1, 2). This family of enzymes mediates the propagation of the intron by cutting intronless genes at the site of intron insertion (reviewed in Ref.3). Like restriction enzymes, homing endonucleases cleave double-stranded DNA with high specificity in the presence of divalent metal ions. However, they differ from restriction endonucleases in their recognition properties and structures, as well as in their genomic location (4). In particular, whereas restriction enzymes have short recognition sequences (3-8 bp), 1 homing endonucleases, despite their small size, recognize long DNA sequences (12-40 bp). They have been classified into four families on the basis of both their sequence motifs and DNA cleavage mechanism (3). to cleave DNA within its recognition sequence and leaves a 4-bp overhang presenting a 3Ј-hydroxyl terminus (5, 6). The enzyme displays a low turnover, probably because of its strong affinity for one of the products of the cleavage reaction (7).The interaction of homing endonucleases with their substrates raises an interesting question common to all the gene-regulatory proteins, namely: how can a small protein specifically recognize and modify a long DNA sequence? Understanding the molecular basis of such a mechanism is essential for elucidating many aspects of cellular control and is a prerequisite in any rational drug design program. It is clearly established that local and global DNA structural features that are highly sequence-dependent, play a primary role in the dynamics of protein-DNA recognition (8). Sequence recognition would...

The present report provides evidence that thymine dimerization can be UVA photosensitized at a tetranucleotide, 5'-TATT-3', by a 7-methyl-pyrido(3,4-c)psoralen monoadduct in DNA. The efficiency of the photoprocess depends on the tetranucleotide flanking sequences. These results demonstrate that one DNA lesion can originate the contiguous formation of a second type of lesion and emphasize the sequence-specific response to interaction of drugs with DNA. Results are related to the sensitivity of DNA to 1,10-phenanthroline-cuprous ion complex nucleolytic activity and discussed in terms of the major role of local deformability of DNA in interaction with ligands.