DNA-DNA INTERSTRAND CROSS-LINKING BY CIS-DIAMMINEDICHLOROPLATINUM(II) - N7(DG)-TO-N7(DG) CROSS-LINKING AT 5'-D(GC) IN SYNTHETIC OLIGONUCLEOTIDES

The nucleotide sequence specificity of the DNA-DNA interstrand cross-linking reaction of cis-diamminedichloroplatinum(II) (cis-DDP) was studied in synthetic oligonucleotides. Of six self-complementary DNAs tested, only those containing the central sequence 5'-d(GC) formed appreciable interstrand cross-linked product, as assayed by denaturing polyacrylamide gel electrophoresis (DPAGE). The nucleotide connectivity of the interstrand cross-link was defined by sequence random oxidative fragmentation followed by DPAGE, revealing that dG residues on opposite strands at the sequence 5'-d(GC) were connected to one another. The covalent structure of the cross-link was established following hydrolysis of the phosphodiester backbone of a structurally homogeneous sample of a cis-DDP interstrand cross-linked DNA tetradecamer. HPLC analysis of the hydrolysate returned all of the deoxynucleoside residues from the starting DNA except for two deoxyguanosine residues. Also returned was diammine-bis-[N7-(2'-deoxyguanosyl)]platinum, identified by a combination of spectroscopic methods, and comparison to a synthetic authentic sample. This study directly establishes that cis-DDP forms interstrand crosslinks at the duplex sequence 5'-d(GC), linking deoxyguanosine residues on opposite strands at N7 through a bridging platinum atom. Computer simulation of the interstrand cross-linked product using molecular mechanics energy minimization and molecular dynamics revealed significant structural reorganization at the site of the cross-link including a ca 40 degrees angle between the platinated guaninyl residues, which propagated to adjacent residues by base stacking to yield duplexes bent by some 30 degrees toward the major groove.