**3. BrdU sensitized formation of interstrand crosslinks (ICL) in mismatched (wobble) DNA duplexes**

In addition to increasing strand break generation by ionizing radiation, the presence of bromouracil induces formation of DNA interstrand crosslinks (ICL). This process occurs in single stranded regions within double stranded DNA (*i.e.*, in scDNA) and requires the presence of B-DNA (Dextraze *et al.*, 2007; 2009). Although anticipated, the generation of ICL during radiosensitization with BrdU has not been demonstrated experimentally prior to our work (Cecchini *et al.*, 2005a). Formation of intra-strand crosslinks has been reported in UVBirradiated BrdU-substituted synthetic DNA (Zeng & Wang, 2006) and in cells (Zeng & Wang, 2007). However, the generation of DNA ICLs by ionizing radiation has been largely ignored in favor to studies on double-strand breaks and their repair. At least part of the problem is technical; it is difficult to detect and quantify ICLs when the same agent forms both strand breaks and ICL, because the analysis of ICL generally involves a denaturing step. Apart from multiple damage events that may cause the disappearance of ICLproducts due to DNA backbone cleavage at nucleotides located between the ICL-site and the radiolabel, another factor that obstructs DNA-ICL detection is that ICLs may decompose under prolonged irradiation, especially in the presence of O2 (Ding & Greenberg, 2007).

oligonucleotides hybridized to complementary or semi-complementary ones with five mismatched bases: AAT(orBU)AA, we have shown that strand breaks are specific for B-DNA, whereas A-DNA only undergoes formation of alkali-labile DNA lesions (Dextraze *et al.*, 2007). Piperidine-sensitive lesions are observed exclusively at the site of BrdU substitution. Generally, the cleavage reaction is a confluence of at least three factors: e<sup>−</sup>aq capture, forward electron transfer and charge recombination. The strand break positions migrate along the DNA strand, however, there is a clear preference for the dA 5'-flanking BrdU. Similarly, 5'-dABrdU-sequence preference has been observed in UV-induced BrdU DNA cleavage (Chen *et al.*, 2000). This is to be expected since in B-DNA the 5' proximal 2' deoxyribose would be the ultimate H-atom donor to the uracil-5-yl radical, whereas in A-DNA conformational (spatial) restrictions render the same 5'dA 2'-deoxyribose a lessaccessible H-donor. Therefore, in A-DNA the uracil-5-yl radical is more likely to abstract Hatoms from other donors (bases), thus oxidizing proximal to the BrdU-site bases which

Sequence-preferential strand break formation was examined in a series of 25-mer scDNA encompassing a central 1- or 5-mer mismatched site with BrdU incorporated (brominated strand) in a purine 5'd(AABrdUAA), 5'd(GGBrdUGG), or a pyrimidine 5'd(ATBrdUTA) environment, and the semi-complimentary (non-brominated) strand contained any of the sequences: 5'd(AATAA), 5'd(CCCCC), 5'd(GGGGG), or 5'd(ATTTA) (twelve permutations in total) (Dextraze *et al.*, 2007; 2009). While there was no significant change between the strand-break yields in ssDNA and typically there were no changes in strand breaks produced on each strand (*i.e.*, brominated *vs*. nonbrominated), two wobble sequence permutations derived from the above pattern: d(GGBrdUGG)^d(GGGGG) and d(ATBrdUTA)^d(ATTTA) produced more breaks on the brominated strand, whereas the generation of breaks was enhanced in the non-brominated strand in the combinations: d(GGBrdUGG)^d(AATAA), d(GGBrdUGG)^d(ATTTA), and d(AABrdUAA)^d(GGGGG). Similarly irradiation of the same scDNA, gave different patterns of interstrand crosslinks

**3. BrdU sensitized formation of interstrand crosslinks (ICL) in mismatched** 

In addition to increasing strand break generation by ionizing radiation, the presence of bromouracil induces formation of DNA interstrand crosslinks (ICL). This process occurs in single stranded regions within double stranded DNA (*i.e.*, in scDNA) and requires the presence of B-DNA (Dextraze *et al.*, 2007; 2009). Although anticipated, the generation of ICL during radiosensitization with BrdU has not been demonstrated experimentally prior to our work (Cecchini *et al.*, 2005a). Formation of intra-strand crosslinks has been reported in UVBirradiated BrdU-substituted synthetic DNA (Zeng & Wang, 2006) and in cells (Zeng & Wang, 2007). However, the generation of DNA ICLs by ionizing radiation has been largely ignored in favor to studies on double-strand breaks and their repair. At least part of the problem is technical; it is difficult to detect and quantify ICLs when the same agent forms both strand breaks and ICL, because the analysis of ICL generally involves a denaturing step. Apart from multiple damage events that may cause the disappearance of ICLproducts due to DNA backbone cleavage at nucleotides located between the ICL-site and the radiolabel, another factor that obstructs DNA-ICL detection is that ICLs may decompose under prolonged irradiation, especially in the presence of O2 (Ding & Greenberg, 2007).

results in alkali-labile site formation.

(see below).

**(wobble) DNA duplexes** 

Therefore, ICL detection and quantitation implies careful selection of the irradiation conditions. In our experience, the presence of a mild •OH-radical scavenger (20 mM EDTA) sufficiently protects against ICL destruction, and even enhances ICL yields up to 1 kGy irradiation dose. A typical ICL- migration pattern observed in agarose denaturing gel electrophoresis of 32P-labeled scDNA samples containing different mismatched regions and subjected to -irradiated (750 Gy) is shown in Fig. 2. The figure exemplifies the fact that, depending on the mismatched-sequence context of the incorporated BrdU, ICL-DNA segments are formed, which differ in their length, structure and yield. Comparative analyses of ICL yields and various electrophoretic-band patterns depending on DNA structure are presented in (Dextraze *et al.*, 2009). Although, no ICL chemical structure identification has been done yet, quantitative data show that d(AABrdUAA)^d(GGGGG) and d(GGBrdUGG)^d(CCCCC) bulge patches are the least prone to crosslink formation in DNA wobbles, while efficient crosslinking takes place in T-enriched bulge structures, *e.g*. d(GGBrdUGG)^d(ATTTA) and d(ATBrdUTA)^d(ATTTA). The calculated total ICL radiation yield (G) in the later sequences (*i.e*. including all ICL bands) is in the range of (1-4) x10-8 mol.J-1. Taking into account that G(e¯aq) = 2.75x10-7 mol J-1 it follows that the formation of interstrand crosslinks in BrdU-scDNA is an event that occurs at least once with every ten solvated electrons produced. Ding and Greenberg (2007) reported -radiolysis production of ICL in unsubstituted dsDNA and identified the structure as T[5m-6n]A (Fig. 6B). Under their irradiation conditions the estimated G-value of the single ICL is ~ (3-4) x10-4 nmol.J-1. These data underline that BrdU-sensitized ICL formation in mismatched scDNA duplex is much more efficient (> 104 - fold) than in normal dsDNA.

Fig. 2. Sequence-dependent BrdU-radiosensitized formation of ICL in wobble scDNA, as seen by the different electrophoretic mobility of the ICL bands. The central quintet sequence of the brominated strand is shown at the bottom; the opposite strand comprises d(A2TA2) or d(C)5 as shown on the top; there is a 5-b.p. central mismatch in all samples, except for lane-3 (panel A) where a single BU^T mismatch is present. The figure exemplifies the role of mismatched sequence (length and composition) on the ICL nature and yields; the individual chemical structures, however have not been yet determined. For a tentative ICL assignment, see Dextraze M.-E., *et al.*, (2009); for recently identified ICL structures, see Fig. 6.

DNA Radiosensitization: The Search for Repair Refractive

A B

Lesions Including Double Strand Breaks and Interstrand Crosslinks 423

Fig. 4. Electrostatic potentials surrounding 10-mer 3D-models of (DNA)2 and DNA-PNA duplexes, calculated using Sybyl modeling interface and dielectric constant, D = 4. Colorcodes as indicated: electronegative potentials -3.5 eV, -1.7 eV (dark blue and yellow) and electropositive +0.2 eV (red). (A) Symmetric electronegative potential surfaces along the two strands of the DNA duplex expand over all backbone atoms (blue) and the two grooves

enveloped by a negative surface (-3.5 eV). The resultant dipole momenta (green vectors) are 19.2 D and 60.4 D for the DNA-DNA and PNA-DNA duplexes, respectively. In the latter case it is oriented diagonally from PNA to DNA strand and can be a driving force for e¯aq

We have studied hybridization of DNA oligonucleotides with PNA, where PNA bear (or not) N- or C-terminal amino groups (-NH2, lysine, or methylmorpholinium) (Fig. 3, Gantchev *et al.*, 2009). After -irradiation (typically 750 Gy) of PNA-DNA heteroduplexes, those with PNA containing free amino group ends formed ICLs (Fig. 5). The multiple bands in each lane represent different crosslinked products and match the number of available amino groups in each heteroduplex. The ICL-formation efficiency is high, G = (5-8) x10-8 mol.J-1. This G-value even exceeds the ICL yields observed after irradiation BrdUsubstituted wobble DNA under identical conditions. Using selective scavengers it was shown that ICL formation in PNA-DNA heteroduplexes strongly depends on the availability of solvated electrons (e¯aq), but proceeds only with a concomitant presence of •OH radicals (Gantchev *et al.*, 2009). Thus, it appears that PNA-DNA ICLs arise in a concerted free radical mechanisms resembling those involved in DNA multiply damaged sites (MDS). By hybridizing 12-mer PNAs with shorter (11-mer), or longer (up to 16-mer) complementary oligo-deoxyribonucleotides thus creating unpaired (single-stranded) regions (deletions and overhangs) at one, or both duplex ends we compared sequence effects on the cross-linking reaction (*e.g.* dT vs. dA termini), the susceptibility of duplex ends to radiation damage, *etc*. The 3'- and 5'- DNA terminal dT nucleotides proved to be of most importance for the efficient ICL formation. Since hydrolysis of N-glycosidic bonds in -

(yellow). (B) Asymmetric isopotential surfaces around PNA-DNA duplex. The electropositive/neutral PNA backbone region (red) extends over the minor and major groove atoms (0 - 0.2 eV at distances ~ 0.1 Å), but the DNA backbone atoms remain

interaction with accessible PNA backbone and groove atoms.
