DNA INTERSTRAND CROSS-LINKING BY FORMALDEHYDE - NUCLEOTIDE-SEQUENCE PREFERENCE AND COVALENT STRUCTURE OF THE PREDOMINANT CROSS-LINK FORMED IN SYNTHETIC OLIGONUCLEOTIDES

The nucleotide sequence specificity of the DNA interstrand cross-linking reaction of formaldehyde and the covalent structure of the predominant cross-link were established in synthetic DNA duplexes. A panel of synthetic DNAs was exposed to aqueous formaldehyde and analyzed by denaturing polyacrylamide gel electrophoresis (DPAGE). Those containing the sequence 5'-d(AT) afforded a relatively abundant interstrand cross-linked product. For one of these products, sequence random fragmentation using iron(II) EDTA/hydrogen peroxide/ascorbic acid and subsequent DPAGE analysis revealed the cross-link to bridge deoxyadenosine residues at the sequence 5-d(AT). Enzymatic digestion of the sugar-phosphate backbone followed by HPLC resolution of the resulting residues afforded, in addition to the common deoxynucleotides, a substance identified by a combination of chemical and spectroscopic studies as bis(N6-deoxyadenosyl)methane. The potential roles of monoadduct formation and progression of monoadducts to cross-links in the mechanistic origin of the selectivity for 5'-d(AT) are discussed. It is speculated that preferential monoadduct formation in AT-rich DNA accounts for formation of monoadducts of deoxyadenosine and that the combination of helical twist and propeller twist, both of which act to orient the amino groups at 5'-d(AT) for cross-linking, are responsible for selection of the sequence 5'-d(AT). It is noted that negative propeller hypertwisting is common to the best available structural model of several interstrand cross-linked DNAs and may therefore play a general role in determining which nucleotide sequences are susceptible to formation of interstrand cross-links,