Sequence Specificity of DNA-DNA Interstrand Cross-Link Formation by Cisplatin and Dinuclear Platinum Complexes

The sequence specificity of interstrand cross-links induced in DNA by mononuclear and dinuclear platinum complexes in a 49-base-pair DNA duplex has been determined directly. This new assay takes advantage of the fact that 3'-->5' exonuclease digestion of randomly platinated DNA produces a pool of fragments of different lengths. This treatment allows identification of the spectrum of adducts impeding the exonuclease scission. Interstrand cross-linked adducts produce fragments that may remain complementary in the proximity of the binding site. As a result, these fragments may act as primer templates for extension upon subsequent treatment with a DNA polymerase. This extension increases the size of the oligonucleotide fragments, which may be evidenced by a more slowly migrating band on a sequencing gel. Concomitantly, the original band corresponding to the digested cross-link decreases in intensity. Therefore, comparison of a sequencing gel after digestion only and after the "digestion-extension" treatment should show the disappearance, or diminished band intensity, of only those fragments with interstrand cross-links. This approach was applied to the analysis of DNA interstrand cross-links formed by cis-[PtCl2(NH3)2] (cis-DDP) and [(trans-PtCl(NH3)2)2H2N(CH2)4NH2]Cl2. Cis-DDP was confirmed to form interstrand cross-links at d(GC) sequences but, interestingly, interstrand cross-links predominated in a sequence GCGG, with possible 1,3-intrastrand but no 1,2-intrastrand cross-links forming. The dinuclear compound formed 1,2, 1,3, and 1,4 DNA interstrand cross-links between guanines on opposite strands. In 1,3 and 1,4 cross-links, the guanines are separated by one and two base pairs, respectively, whereas a 1,2 cross-link is formed from guanines on neighboring base pairs.(ABSTRACT TRUNCATED AT 250 WORDS)