**2. DNA-structure/conformation dependent BrdU-sensitized formation of strand breaks**

A crucial difference between the radiosensitization of single and double stranded DNA by BrdU was not known until our work with purified *semi*-complementary (mismatched) DNA showing the single stranded specificity of BrdU-induced DNA damage (Cecchini *et al.*, 2004). In parallel experiments, using BrdU-substituted (or not) single-stranded (ssDNA), double-stranded (dsDNA) and mismatched (wobble) semi-complementary (scDNA) DNA we have found that BrdU efficiently sensitizes single stranded BrdU-substituted (brominated) oligonucleotides, but not when these are hybridized to completely complementary oligonucleotides to form normal dsDNA duplexes. We estimate that BrdU radiosensitization efficiency in dsDNA drops up to 20-fold compared to that in ssDNA. Comparative measurements of radiolytic loss of the bromine atom in ssDNA *vs*. dsDNA likewise indicate that this process is greatly suppressed in dsDNA (Cecchini *et al.*, 2004, 2005a). In mismatched, scDNA duplexes, strand brakes are formed in loci encompassing nucleotides surrounding BrdU. However, high efficiency single strand break formation takes place on the brominated strand, or on the opposite, non-brominated strand but have not been detected on both, suggesting that they are mutually exclusive events (Hunting *et al.*, unpublished; Cecchini *et al.*, 2005b). Experiments performed with scDNA bearing variable number of mismatches (from one to five) and containing a single BrdU substitution gave qualitatively similar results. The radiation dose-response for strand break formation was linear for both the brominated and the opposite, non-brominated strand within the single-stranded regions of a standard model scDNA containing a bulge formed by up to five mismatched bases (Dextraze *et al.*, 2007). Interestingly, UVB-irradiation of BrdU-substituted DNA also demonstrated DNA-structure specificity, but in this situation BrdU greatly enhances breakage of only the brominated strand in dsDNA, or either the brominated or the non-brominated strand in the case of scDNA (Cecchini *et al.*, 2005b; Chen *et al.*, 2000). The different effects initiated by radiolysis and photolysis, especially with BrdU-dsDNA, underline the role of DNA structure-conformation properties in solution as a prerequisite for the initial electron-capture by BrdU and/or the propagation of an excess-electron along the polymer after gamma-irradiation.

The importance of DNA structure during sensitization by BrdU is further demonstrated by comparison of the damages induced in A- and B-form DNA. Using brominated 25-mer

DNA Radiosensitization: The Search for Repair Refractive

much more efficient (> 104 - fold) than in normal dsDNA.

Lesions Including Double Strand Breaks and Interstrand Crosslinks 421

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

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.

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 results in alkali-labile site formation.

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 (see below).
