Thermal stability of DNA adducts induced by cyanomorpholinoadriamycinin vitro

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Abstract
The Adriamycin derivative, cyanomorpholinoadrla-mycln (CMA) was reacted with DNA in vitro to form apparent Interstrand crosslinks. The extent of Interstrand crosslink formation was monitored by a gel electrophoresis assay and maximal crosslinking of DNA was observed within 1 hr with 5 μM of drug. The interstrand crosslinks were heat labile, with a midpoint melting temperature of 70°C (10 min exposure to heat) In 45% formamlde. When CMA-induced adducts were detected as blockages of λ-exonuclease, 12 blockage sites were observed with 8 being prior to 5′-GG sequences, one prior to 5′-CC, one prior to 5′-GC and 2 at unresolved combinations of these sequences. These exonuclease-detected blockages reveal the same sites of CMA-lnduced crosslinking as detected by in vitro transcription footprinting and primer-extension blockages on single strand DNA, where the blockages at 5′-GG and 5′-CC were Identified as sites of Intrastrand crosslinking and the 5'-GC blockage as a probable site of Interstrand crosslinking. The thermal stability of both types of crosslink (10 min exposure to heat) ranged from 63–70°C at individual sites. High levels of adduct were detected with poly (dG-dC) but not with poly (dl-dC). These results suggest adduct formation involving an aminal linkage between the 3 position of the morphollno moiety and N2 of guanlne.