DNA HAIRPIN FORMATION IN ADDUCTS WITH PLATINUM ANTICANCER DRUGS - GEL-ELECTROPHORESIS PROVIDES NEW INFORMATION AND A CAVEAT

The duplex form of the self-complementary oligonucleotide, T(2)G(3)G(4 )G(5)T(6)A(7)C(8)C-(9)C(10)A(11)T(12)), was treated with cis-Pt(A2)Cl2 anticancer drugs(A2=(NH3)2 or en(ethylenediamine)). Previous NMR repo rts of the Pt(en) reaction revealed one hairpin-like product with plat ination at two of G(4), G(5), or A(7) but not at G(3). To resolve thes e issues, 3'-P-32-end-labeling of reaction mixtures was examined. Gel electrophoresis gave one pronounced product band (autoradiographic det ection) with hairpin-like mobility for both drugs. However, in conflic t with the NMR studies, this product has a G(3),G(4) intrastrand cross link (DNA sequencing methods). Furthermore, with each drug, 5'-P-32-en d-labeling and gel electrophoresis gave two significant comparable hai rpinlike product bands: one for the G(3),G(4) crosslinked adduct ident ified as the exclusive product by 3'-P-32-end-labeling and the second for a G(4),G(5) crosslinked adduct consistent with NMR studies. To res olve these issues, the Pt(en) reaction was subjected to exhaustive 5'- end-labeling with nonradioactive ATP. The G(3),G(4) adduct identified as the exclusive product by 3'-P-32-end-labeling was found by UV-visua lization to constitute only 4% of the product. The major product (96%) was the G(4),G(5) crosslinked adduct. From gel electrophoresis under denaturing conditions, the G(3),G(4) adduct exists in the denatured st ate to a much greater extent than the G(4),G(5) adduct. Clearly, durin g both types of enzymatic labeling, the minor product (probably as the denatured form) was labeled at much higher efficiency, suggesting tha t caution should be exercised in interpreting the increasingly widely used P-32/gel electrophoresis methods. In a GGG sequence, the N7 of th e central residue, G(4), is the most nucleophilic site, and we found t hat the monofunctional complex, [chloro(diethylenetriamine)platinum(II )] chloride, preferentially attacks this site. A(7) binding was shown to be insignificant both by the diethyl pyrocarbonate reaction and by enzymatic digestion, which reveals only G,G crosslinking. These result s suggest that an initial G(4) monoadduct forms a 1,2 crosslink to the 3'-end G(5) much more favorably than to the 5'-end G(3) and that 1,2 G,G crosslinking is much more favorable than 1,4 G,A crosslinking in e ither direction. Relatively stable hairpins such as the G(4),G(5) addu ct described here could explain features of the P-31 NMR spectrum obse rved on treating polymeric DNA with Pt anticancer drugs and could stab ilize cruciforms in palindromic regions. The latter possibility is dis cussed in the light of the recent discoveries on the structure-specifi c recognition protein and its partial sequence homology with the high mobility group protein-1, a species known to recognize cruciforms [Bru hn, S. L.; Pil, P. M.; Essigman, J. M.; Housman, D. E.; Lippard, S. J. Proc. Natl. Acad. Sci. U.S.A. 1992, 89, 2307. Pil, P. M.; Lippard, S. J. Science 1992, 256, 234].