**2.2.2 Carcinogens as DNA destabilizing agents**

DNA interaction of carcinogen, adduct formation and their repair processes are widely studied using carcinogens from environmental and tobacco smoke. Some of them have the ability to destabilize the DNA helix: BPDE ((+/-)-*anti-*benzo[*a*]pyrene-7,8-dihydrodiol-9,10 epoxide) and 4-OHEN (4-hydroxyequilenin-O-quinone) (Figure 4).

The smoke carcinogen benzo[*a*]pyrene (BaP) is metabolized into several enantiomers of BPDE that covalently bond the exocyclic NH2 group of guanines to form a bulky adduct in the minor groove of the DNA helix, resulting in its destabilization (Zou & Van Houten, 1999). Due to the orientation of the reactive epoxide group on asymmetric carbons, several enantiomers are produced. The most carcinogenic is 10S(+)-*trans*-*anti*-BPDE N2-dG adduct followed by the stereo-isomeric 10R(+)-*cis*-*anti*-BPDE-N2-dG adducts. Covalent bonding to DNA is associated with base-displaced intercalation where the bulky adduct prevents the hydrogen bonding of the amino group of guanine with the opposite cytosine. This results in

DNA Helix Destabilization by Alkylating Agents: From Covalent Bonding to DNA Repair 103

decrease in the melting temperature (Tm) of a 11-bp oligonucleotide, with the magnitude of the negative Tm values being lower when the adduct is located at 1 or 2-bp from the end of the 11-bp DNA (-6 to -9°C) then when it is located in its medium part (positions 4 to 8) with up to a -21 to -27°C decrease of Tm. Similarly, the stereoisomeric orientation of the 4-OHEN adduct affects the base-stacking, groove sizes and subsequent distortions and is also crucial for

Psoralen is a chemotherapeutic agent known to cause DNA inter-strand crosslinks (ICLs) upon absorption of two photons from UVA irradiation at 365 nm, preferentially at 5′-TA and to a lesser extend at 5′-AT dinucleotides. This activity was the basis for use of psoralen and UVA exposure (PUVA therapy) to treat cutaneous diseases like psoriasis, vitiligo, atopic dermatitis or cutaneous T cell lymphomas. However, such treatment increased the risk of squamous and basal cell carcinomas (Teicher, 1996). Psoralen-induced ICLs are classically used models for DNA repair of ICLs. The psoralen derivative 4'-(hydroxymethyl)-4,5',8 trimethylpsoralen (HMT) (Figure 4) evidenced DNA destabilization by mono-addition of a

psoralen residue to both thymines (one on each strand) of 5'-GGGTACCC sequence.

Acronycine is a natural alkaloid extracted from the bark of an Australian ash scrub that presented interesting antitumor activities but was poorly soluble and, consequently, too toxic in first clinical trials. The discovery of an unstable acronycine epoxide opened the way to the rational drug design of S23906-1 (Figure 4), that appeared to be a highly active compound (Guilbaud et al., 2001) with an original mode of action (David-Cordonnier 2002; 2005; Depauw et al., 2009) and consequently entered phase I clinical trials in 2006. As for the clinically used drug Ecteinascidine 743 (ET-743, Trabectedin, Yondelis TM from Pharmamar), S23906-1 alkylates the exocyclic NH2 group of guanines in the minor groove. But, in contrast with ET-743, S23906-1 does not reinforce the stability of the ds-DNA helix but destabilizes it, generating portions of ss-DNA (David-Cordonnier et al., 2005; Depauw et al., 2009). Various spectral and biochemical approaches convinced with this conclusion. Indeed, classical DNA melting temperature studies evidenced a strong decrease of the Tm values upon alkylation with S23906-1 or other biologically active benzo-acronycine derivatives. Similarly, spectral analysis of the ratio of fluorescence properties of picogreen (a ds- and ss-DNA interacting dye) and BET (a ds-DNA specific dye) evidenced an increase of picogreen *vs*. BET fluorescence which enlightens the generation of single-stranded portions of the DNA upon S23906-1 alkylation. Biochemical approaches like digestion of the alkylated DNA by singlestrand specific nuclease S1 and electrophoretic mobility shift assays (EMSAs) confirmed the opening of the DNA. The destabilization was relatively wide since mapping with nuclease S1 evidenced locally opened DNA portions within a 117 bp DNA fragment alkylated by S23906-1 whereas EMSAs, performed with oligonucleotides as long as 24 bp, evidenced fully single-stranded alkylated oligonucleotides in the presence of S23906-1 or derivatives

DNA adducts are critical lesions for cell proliferation and survival. Single or multiple DNA repair machineries could be implicated in the removal of these damages, as for example

the extent of DNA destabilization (Kolbanovskiy et al., 2005).

**2.2.3 Psoralen derivatives** 

**2.2.4 Benzo-acronycine derivatives** 

(David-Cordonnier et al., 2005; Depauw et al., 2009).

**3. Repair processes for DNA destabilizing lesions** 

a base-flipping where the (+)-*anti*-B[*a*]P-N2-dG bulky adduct is located in the minor groove and the opposite cytosine is positioned in the major groove (Cosman et al., 1993). The precise orientation of this highly carcinogenic 10S(+)-*trans-anti*-B[*a*]P-N2-dG adduct depends on the sequence surrounding the target guanine (Cai et al., 2010). DNA is untwisted at 5'- CGG\*C sites where a large bend is induced in the DNA helix, but not at 5'-CG\*GC sequences where, conversely, DNA helix is destabilized in its portion orientated 5' to the lesion (Rodríguez et al., 2007). Such differences result in different protein/DNA recognition and repair activities (see 3.4). Thermal destabilization was also observed using 14R(+)-*transanti*-DB[*a*,*l*]P-N2-dG adduct (Zheng et al., 2010) or *14S*(-)-*trans-anti*-DB[*a,l*]P-N6-dA adducts whereas *14R*(+) isomer stabilizes the ds-DNA (Cai et al., 2011).

Fig. 4. 3D orientation of (+)-*anti-*BPDE [mmdbId:52106] and the psoralen derivative HMT [mmdbId:52343] and structure of some DNA alkylators that destabilize the DNA helix.

The hormone-derived genotoxic compound, 4-OHEN, derives from equilin and equilenin, two equine oestrogens present in hormone substitution therapies used to prevent the uncomfortable effects of menopauses but are also thought to increase breast cancer incidence in the population of hormonally-treated women (Rossouw et al., 2002). Its orthoquinone form is cytotoxic and genotoxic (Pisha et al., 2001) through the formation of bulky DNA lesions at dA, dC and dG (but not at T residues) (Kolbanovskiy et al., 2005) which were detected in both cell culture and breast cancer biopsies from patients treated with hormone substitution therapies (Embrechts et al., 2003). 4-OHEN derived from the intermediate catechol 4-hydroxyequilenin which was generated from a rapid conversion of both equilin and equilenin in the organism to four stereo-isomers differently affecting the 3D-structure of the DNA helix (Ding et al., 2007). For adducts on cytosine, the *syn*- or *anti*conformations of the bulky rings of 4-OHEN point along the major or the minor groove (Ding et al., 2005). Interestingly, alkylation at dA or dC residues is associated with a strong decrease in the melting temperature (Tm) of a 11-bp oligonucleotide, with the magnitude of the negative Tm values being lower when the adduct is located at 1 or 2-bp from the end of the 11-bp DNA (-6 to -9°C) then when it is located in its medium part (positions 4 to 8) with up to a -21 to -27°C decrease of Tm. Similarly, the stereoisomeric orientation of the 4-OHEN adduct affects the base-stacking, groove sizes and subsequent distortions and is also crucial for the extent of DNA destabilization (Kolbanovskiy et al., 2005).
