Intrastrand crosslinks

The different possible adducts formed between mitomycin C and DNA have been isolated by degradation of DNA after in vitro alkylation/crosslinking reactions and structurally characterized. Monoadduct 21 (Scheme 11.3), derived from alkylation at C-l only [53], and monoadducts 22 [54] and 23 [55, 56] (derived from C-10 alkylation by 16 at N-7 or N-2 of guanine, respectively) have been isolated, together with bisadducts 24 [57] and 25 [58], derived from interstrand and intrastrand crosslinks, respectively, and adduct 26 [59], formed by addition of a molecule of water to C-10 instead of the second guanine. All of these adducts have also been isolated from DNA after in vivo crosslinking [60, 61]. 

Binding of cisplatin to the neighbouring bases in the DNA disrupts the orderly stacking of the purine bases when it forms a 1,2-intrastrand crosslink, it bends the DNA helix by some 34° towards the major groove and unwinds the helix by 13°. These cross-links are believed to block DNA replication. 

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. 

Data taken from N. Poklar, D. S. Pilch, S. J. Lippard, E. A. Redding, S. U. Dunham, and K. J. Breslauer, Influence of Cisplatin Intrastrand Crosslinking on the Conformation, Thermal Stability and Energetics of a 20-mer DNA Duplex, Proc. Natl. Acad. Sci. USA, 93, 7606-76U (1996). 

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...

The formation of intrastrand crosslinks on GG and AG base sequences in DNA could be deduced from the presence of the adducts ds-Pt(NH3)2(d(pGG)) and cis-Pt(NH3)2(d(pAG)) in digests of platinated DNA. However, no indications for the presence of GA adducts could be found, and therefore crosslinks with a GA sequence are unlikely. 

Recently, it could be established that in living cells the same types of Pt-adducts are formed as in DNA treated with cisplatin in vitro120,121. Also the strong preference of cisplatin to form intrastrand crosslinks on GG base sequences is found upon interaction with DNA in vivo120. ... 

The question which of the induced Pt-DNA adducts is (are) responsible for the antitumor activity is still unanswered. Many investigators have tried to correlate interstrand DNA and DNA-protein crosslink formation with the cytotoxic action of cisplatin. However, conflicting results were obtained18,50 Until now a biological role inside the cell has been indicated for only one type of Pt-DNA adduct. Brouwer et al.48 showed that cisplatin can induce base-pair substitutions in E. coli bacteria at GAG and GCG base sequences. This strongly suggests that the intrastrand crosslink of cisplatin on GBG is responsible for this effect. 

Patients, suffering apparently from the same type of tumor, often respond differently to Pt-chemotherapy. Therefore, a research program has been started on the Pt-adduct formation and repair in the DNA of the nucleated peripheral blood cells of these patients. Figure 22 shows the number of intrastrand crosslinks on pGG sequences per 109... 

Fig. 22. Numbers of intrastrand crosslinks to GG-units in DNA isolated from white blood cells of two different patients at various time intervals after the onset of the cisplatin infusion (indicated by arrows).

NMR Methods can be used to obtain dynamic, structural, and thermodynamic information on solutions. Until recently, all evidence has pointed to the intrastrand crosslink having a head-to-head (HH) form with the guanine bases oriented in the same direction (Fig. 3). We shall discuss the fea-... 

The reaction of [(S,/ ,R,S)-BipPt(H20)2]2+ with d(GpG) also unexpectedly yielded two products of comparable abundance. One adduct was characterized to be the normal HHl form. The G H(8) shift pattern of this HH form, namely 5 -G H(8) upfield and 3 -G H(8) downfield, is the same as that found for ds-Pt(NH3)2(d(GpG)), suggesting that these two adducts have similar hydrogen bonding and base canting. The 31P-NMR signal at -2.8 ppm is also a common feature of HH adducts. The second adduct, determined to be an HT conformer, has several unique spectral features. Of particular note, the relatively upfield shifts of both G H(8) signals (7.91 and 7.77 ppm) and an upfield-shifted 31P-NMR signal (-4.6 ppm) of the HT conformer are unprecedented for a major conformer of an adjacent G -G intrastrand crosslinked species. 

Hypothesis III The known force fields, designed to reproduce typical DNA structures, fail to meet the challenge presented by accommodating NMR restraints from a distorted duplex with an intrastrand crosslink. 

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