Strand scission

FIGURE 12.4 Maxam-Gilbert sequencing of DNA cleavage at purines uses dimethyl sulfate, followed by strand scission with piperidine. 

Cleavage at A or G If the DNA is first treated with acid, dimethyl sulfate methylates adenine at the 3-position as well as guanine at the 7-position (not shown). Subsequent reaction with OH and piperidine triggers degradation and displacement of the methylated A or G purine base and strand scission, essentially as indicated here for reaction of dimethyl sulfate with guanine. 

Oxidative nucleobase modifications leading to strand scission 98CRV1109. 

HydroxymethylquinoxalLne (and a corresponding ester, methyl 2-quinoxaline-carboxylate) have been found to induce significant single-strand scission of DNA by additive-free irradiation at 365... 

Cooperative work with Prof. Nobuo Tanaka (Professor Emeritus of the University of Tokyo) in 1969 showed the mechanism of action of bleomycin to involve DNA strand-scission. The difficult total synthesis of bleomycin was accomplished (1981) in cooperation with Takita and others, including Hamao s son, Yoji Umezawa. H. Umezawa was very satisfied with the success of this total synthesis, and his sustained enthusiasm for improved bleomycins led to peplomycin (1978 used clinically since 1981) and libro-mycin(1985). 

Flowers, L. Ohnishi, T. Penning, T. M. DNA strand scission by polycylic hydrocarbon o-quinone role of reactive oxygen species, Cu(II)/(I) redox cycling, and o-semiquinone anion radicals. Biochemistry 1997, 36, 8640-8648. 

On the other hand, if the hole flow in DNA could be artificially controlled to deposit at the desired site in DNA, it may enable site-selective oxidation and strand scission of DNA, which is desirable from a therapeutical standpoint. Furthermore, understanding DNA-mediated hole transfer is expected to lead to an additional application in the development of biosensors and bioelectronic devices [9]. Therefore, the regulation of the transfer rate and direction of the hole generated in DNA is of interest from the perspective of using DNA as a building block for electronic devices. 

In vitro studies of DNA interactions with the reactive ben-zo[a]pyrene epoxide BPDE indicate that physical binding of BPDE occurs rapidly on a millisecond time scale forming a complex that then reacts much more slowly on a time scale of minutes (17). Several reactive events follow formation of the physical complex. The most favorable reaction is the DNA catalyzed hydrolysis of BPDE to the tetrol, BPT (3,5,6,8,17). At 25°C and pH=7.0, the hydrolysis of BPDE to BPT in DNA is as much as 80 times faster than hydrolysis without DNA (8). Other reactions which follow formation of physical complexes include those involving the nucleotide bases and possibly the phosphodiester backbone. These can lead to DNA strand scission (9 34, 54-56) and to the formation of stable BPDE-DNA adducts. Adduct formation occurs at the exocyclic amino groups on the nucleotide bases and at other sites (1,2,9,17,20, 28,33,34,57,58). The pathway which leads to hydrocarbon adducts covalently bound to the 2-amino group of guanine has been the most widely studied. 

Xanthine oxidase, a widely used source of superoxide, has been frequently applied for the study of the effects of superoxide on DNA oxidation. Rozenberg-Arska et al. [30] have shown that xanthine oxidase plus excess iron induced chromosomal and plasmid DNA injury, which was supposedly mediated by hydroxyl radicals. Ito et al. [31] compared the inactivation of Bacillus subtilis transforming DNA by potassium superoxide and the xanthine xanthine oxidase system. It was found that xanthine oxidase but not K02 was a source of free radical mediated DNA inactivation apparently due to the conversion of superoxide to hydroxyl radicals in the presence of iron ions. Deno and Fridovich [32] also supposed that the single strand scission formation after exposure of DNA plasmid to xanthine oxidase was mediated by hydroxyl radical formation. Oxygen radicals produced by xanthine oxidase induced DNA strand breakage in promotable and nonpromotable JB6 mouse epidermal cells [33]. 

Of wider significance was the generation of 2-phenylethyl radical by oxyhemoglobin-mediated oxidation of phenelzine (2-phenylethylhydrazine), which was shown to be more efficient in promoting alkali-labile sites than in producing direct DNA strand scission (Fig. 6) [25]. 

GUoni, L., Takeshita, M., Johnson, F., Iden, C., and Grollman, A. P., 1981, Bleomycin-induced strand-scission ofDNA. Mechanism of deoxyiibose cleavage,/. Biol. Chem. 256 8608-8615. 

Non-sequence-specific or global DNA damage is generally caused by random DNA alkylation, cross-linking or strand scission, the mechanisms and consequences of which have been previously mentioned. 

Giloni L, Takeshita M, Johnson F, Iden C, Grollman AP (1981) Bleomycin induced strand-scission of DNA. Mechanism of deoxyribose cleavage. J Biol Chem 256(16) 8608-8615 Gniazdowski M, Czyz M (1999) Transcription factors as targets of anticancer drugs. Acta Biochim Pol 46(2) 255-262... 

As the quencher is negatively charged, this electron transfer reaction and the subsequent reactions involving the negatively charged SO " radical, are less efficient when the complex binds to DNA than when it remains in solution, nevertheless SO and the oxidised complex oxidise the bases (B) of nucleic acid, eventually leading to strand scissions (see Sect. 5). 

It would be interesting to test with other Rh(III) complexes, whether the direct oxidation of the base (by photo-electron transfer) could also be a primary step responsible for photocleavages. Indeed, as outlined before in Sect. 5, radiation studies have shown that the radical cation of the base can produce the sugar radical, itself leading to strand scission [122]. Moreover base release, as observed with the Rh(III) complexes, can also take place from the radical cation of the base [137]. Direct base oxidation and hydrogen abstraction from the sugar could be two competitive pathways leading to strand scission and/or base release. 

Co(phen) and Co(DIP) have been reported to cleave DNA upon irradiation with UV light (k < 320 nm) [117,147]. As no mechanistic studies were performed, the different reactions leading to strand scissions are not known. Photoreduction of the Co(III) species could constitute the initial step of the reaction pathway. 

CofNHs) also photo-cleaves DNA [117], but in this case, formation of Co (II) and oxidised ligand from the LMCT state could represent an alternative pathway to a direct oxidation, leading to strand scission. 

These results seem to suggest that by irradiation in alkylamines DNA (single stranded) can undergo a selective release of thymine, accompanied by strand scission. This has indeed experimentally been verified. Irradiation of calf thymus DNA in 5% aqueous n-butylamine gave a photoproduct that on heating yields 1-n-butylthymine (81MI1). 

The bleomycins (50) are hardly simple amines, but they do have two NH2 groups and a CONH2 group at the N-terminal domain, as well as potential donor nitrogens in pyrimidine and imidazole, which can complex metal ions." " The complexing of iron to bleomycin" " " has a significant effect on bleomycin-DNA interactions—metal complexes can mediate strand scission—and on alkene oxidation. Both may involve hydroperoxide intermediates." " " " ... 

It has been recently shown that the selective alkylation and strand scission of deoxytetranucleotide d(GTAG)-27 chosen as DNA model, results from the formation of covalent adducts 28 and 29 on the N-7 of guanine and N-3 of adenine with opening of the cyclopropane ring, respectively. Thermal treatment of 28 (90 °G, 5 min) afforded the d(deoxyribose-TAG) 30 with liberation of N-7 alkyl-guanine 31, while treatment of 29 provided the d(GT-deoxyribose-G) 32 and the N-3 alkyladenine 33 [27]. The stabilities of adducts 28 and 29 were tj/2 = 31 h and 3.2 h, respectively therefore, the cleavage reaction of adduct 29 proceeds much faster than that of 28, Eq. (11) [27]. 

Bleomycin intercalates between bases (f d ouble-stranded DNS(34). In this connection, it is interesting that phleomycin which has the dihydrobithiazole moiety, does not intercalate with DNA (33). It causes more single-strand scission than double-strand scission (30). 

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