THERMAL-STABILITY OF DNA ADDUCTS INDUCED BY CYANOMORPHOLINOADRIAMYCININVITRO

The Adriamycin derivative, cyanomorpholinoadriamycin (CMA) was reacted with DNA in vitro to form apparent interstrand crosslinks. The extent of interstrand crosslink formation was monitored by a gel electrophor esis assay and maximal crosslinking of DNA was observed within 1 hr wi th 5 muM of drug. The interstrand crosslinks were heat labile, with a midpoint melting temperature of 70-degrees-C (10 min exposure to heat) in 45% formamide. When CMA-induced adducts were detected as blockages of lambda-exonuclease, 12 blockage sites were observed with 8 being p rior to 5'-GG sequences, one prior to 5'-CC, one prior to 5'-GC and 2 at unresolved combinations of these sequences. These exonuclease-detec ted blockages reveal the same sites of CMA-induced crosslinking as det ected by in vitro transcription footprinting and primer-extension bloc kages on single strand DNA, where the blockages at 5'-GG and 5'-CC wer e identified as sites of intrastrand crosslinking and the 5'-GC blocka ge as a probable site of interstrand crosslinking. The thermal stabili ty of both types of crosslink (10 min exposure to heat) ranged from 63 -70-degrees-C at individual sites. High levels of adduct were detected with poly (dG-dC) but not with poly (dl-dC). These results suggest ad duct formation involving an aminal linkage between the 3 position of t he morpholino moiety and N2 of guanine.