Interstrand crosslinking, DNA

The Assessment of DNA Crosslinking by Alkaline Elution. DNA damage, that is, interstrand crosslinks, DNA-protein crosslinks, and strand breaks, was determined using the alkaline elution technique (7, ). Cells labeled with C-thymidine for 20-24 hr were deposited on a membrane filter and lysed with a detergent-containing solution. An alkaline solution (pH 12.1-12.2) was then slowly pumped through the filter, and fractions were collected to determine the rate of release of DNA from the filter. For assay of crosslinks, the cells were exposed to x-ray at 0 C prior to deposition on the filter. In order to improve quantitation, control cells labeled with H-thymidine and x-irradiated at 0 were mixed with the experimental Relabeled cells prior to deposition on the filters. The elution of H-DNA serves as an internal reference for normalization of the elution of C-DNA. 

Interstrand crosslink DNA-protein crosslink Intrastrand crosslink Figure 2 General crosslinked forms of DNA from reaction with cisplatin. 

Dialysis experiments and DNA thermal denaturation studies of bisimidazoletetrachloroirida-te(III), (250), suggest poor binding of (250) to DNA, with no formation of interstrand crosslinks.422... 

Qu, Y. Scarsdale, N. J. Tran, M. C. Farrell, N. Cooperative effects in long-range 1,4 DNA-DNA interstrand crosslinks formed by polynuclear platinum complexes an unexpected syn-orientation of adenine bases outside the binding sites. J Biol Inorg Chem 2003, 8, 19-28. 

Kasparkova, J. Zehnulova, J. Farrell, N. Brabec, V. DNA interstrand crosslinks of novel antitumor trinuclear platinum complex BBR. Conformation, recognition by HMG-domain proteins and nucleotide excision repair. J Biol Chem 2002, 277, 48076-48086. 

Ultraviolet-induced cleavage of both intra- and interstrand crosslinks involving Pt-G bonds has been observed by Payet and Leng (140). Kane and Lippard (141) have reported that irradiation of DNA modified by cisplatin with UV light (A >300 nm) can induce specific cross-links to HMG1, thought to involve Lys6 in the B-domain, with labilization of a Pt-purine bond. 

If a DNA adduct involves the nitrogen or oxygen atoms involved in base-pairing, and the adducted DNA is not repaired, base substitution can result. Adducts can be small, such as the simple addition of methyl or ethyl groups, or they can be very bulky, owing to reaction with multiringed structures. The most vulnerable base is guanine, which can form adducts at several of its atoms (e.g., N7, C8, O6 and exocyclic N2) (Venitt and Parry, 1984). Adducts can form links between adjacent bases on the same strand (intrastrand cross-links) and can form interstrand crosslinks between each strand of double-stranded DNA. 

Coste F, Malinge J-M, Serre L, Shepard W, Roth M, Leng M, Zelwer C. Crystal structure of a double-stranded DNA containing a cisplatin interstrand crosslink at 1.63°A resolution hydration at the platinated site. Nucleic Acids Res 1999 27 1837-1184. 

Perez C, Leng M, Malinge JM. Rearrangement of interstrand crosslinks into intrastrand crosslinks in cis-diamminedichloroplatinum(II)-modified DNA. Nucleic Acids Res 1997 25 896-903. 

The relatively acidic thiol on serum albumin forms a fairly stable S-nitroso adduct with nitric oxide, which may serve to preserve and carry NO throughout the circulatory system [162,163]. Bacterial toxins released in toxic-shock syndrome induce excessive NO-synthase activity in macrophages. The resulting arterial expansion may induce the cardiovascular collapse associated with toxic shock syndrome [164]. Nitrous acid reacts with DNA to form dG-N2-dG interstrand crosslinks at the sequence 5 CG [165]. NO can also deaminate cytidine [166] and deoxyguanosine [167] and so may function as a mutagen. The rate law for NO reacting with O2 has been measured electrochemically as [168] ... 

Importantly, the ERCCl/XPF structure-specific nuclease has an additional role in the repair of cisplatin adducts besides its function in NER the recombinational repair of interstrand crosslinks (19). ERCCl- or XPF-deficient hamster mutant cell lines are hypersensitive to DNA crosslink agents, much more so than to ultraviolet-induced pyrimide dimers, the classical substrates for NER (20,21). Moreover, co-localization of ERCCl foci and RAD51 foci in response to cisplatin treatment has recently been found and may represent recruitment of ERCCl/XPF to sites of recombination repair (22). Previous studies have shown that BRCAl, involved in homologous recombination repair, also plays a major role in the repair of cisplatin DNA damage (23). 

Wojcik A, Bochenek A, Lankoff A, Risowska A, Padjas A, von Sonntag C, Obe G (2005) DNA interstrand crosslinks are induced in cells prelabelled with 5-bromo-2 -deoxyuridine and exposed to UVC radiation, in preparation... 

Fig. 7. Schematic representation of cisplatin bonding to DNA. (1) monofunctional binding (X = Cr, OH", OH2) (2) interstrand crosslinking (3) protein-DNA crosslinking (4) intrastrand crosslinking between adjacent guanines (5) intrastrand crosslinking between two guanines separated by a third base (6) intrastrand crosslinking at a-AG-unit...

Cyclophosphamide (6.31) is a nitrogen mustard used for cancer treatment (Scheme 6.9). In the body, cyclophosphamide is oxidized to aminal 6.32. Compound 6.32 opens and loses acrolein to form phosphoramide mustard (6.33). Structure 6.33 is a strong bis-electrophile and reacts readily with nucleophiles. In DNA, the nucleophile tends to be N7 of guanine, which is oriented outward into the major groove (Figure 6.6). By reacting twice, 6.33 crosslinks DNA either within the same strand (intrastrand) or across the double helix (interstrand).16... 

Bridgewater LC, Manning FCR, Patierno SR. 1994a. Base-specific arrest of in vitro DNA replication by carcinogenic chromium relationship to DNA interstrand crosslinking. Carcinogenesis 15(11) 2421-2427. 

The rate of formation of cisplatin-DNA adducts was found to be independent of superhelicity [59] and appears to be unaffected by the presence of histones in nucleosomes [29] and in chromatin [77], Therefore, isolated DNA in aqueous solution appears to be a relevant model for kinetic and mechanistic studies of cellular DNA-platination. It was early checked that the cisplatin-DNA adducts were stable for a least three days at 37 °C after their formation [78], There are now a few cases reported of unstable platinum adducts (vide supra) i) monoadducts with the diazapyrenium ligand [52], ii) a cisplatin intrastrand GG chelate rearranging into a GG interstrand crosslink [63], iii) cisplatin GG interstrand diadducts, slowly rearranging into intrastrand ones [65], tv) transplatin intrastrand GNG diadducts rearranging into interstrand crosslinks (J.-M. Malinge and M. Leng, Part 3). 

Because of the importance of the d(G pG ) crosslink, previous studies in which 1,2-intrastrand d(G pG ) crosslinks have been modeled and investigated using NMR spectroscopy are reviewed. Models for interstrand crosslinks are also reviewed, although fewer studies are available [21][22], A number of techniques can be utilized to study the conformation of Pt-DNA adducts, but NMR spectroscopy is the focus of this chapter. There are three types of models for Pt-DNA interactions that contribute to our understanding of the interpretation of NMR results a) Pt complexes with nucle-obases, nucleosides, or nucleotides (abbreviated in this work as c -PtA2G2 A2 = a diamine or two amines, and the G derivative is bound via N(7) in all cases described here) b) Pt adducts with single-stranded DNA (Pt-ssDNA) and c) Pt complexes with double-stranded DNA (Pt-dsDNA). 

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