Effects of spermine and its cytotoxic analogs on nucleosome formation on topologically stressed DNA in vitro.

We investigated the effects of the polyamine spermine and two of its cytotoxic analogs 1,11-bis(ethylamino)-4,8-diazaundecane (BE-3-3-3) and 1,19-bis(ethylamino)-5,10,15-tirazanonadecane (BE-4-4-4-4) on the formation of nucleosomes on negatively and positively supercoiled DNA in vitro. Histones H2A, H2B, H3 and H4 were reconstituted onto DNA to form nucleosomes and the polyamines were added either before or after histone addition. The structural state of the nucleosome was monitored by analyzing the DNA topoisomers that were present after topoisomerase I treatment. Although polyamines induced DNA aggregation to various degrees. high concentrations of topoisomerase I were able to relax the aggregated DNA and the helical pitch was found to be unaltered in the aggregates. When histones were associated with negatively coiled DNA, the polyamine-induced aggregation did not alter nucleosome structure. The induced aggregate did inhibit nucleosomal transitions when examined on positively coiled DNA. BE-4-4-4-4 was most effective and BE-3-3-3 least effective. These analogs were also extremely effective in inhibiting histone deposition onto DNA. A potential mechanism for the action of these analogs is both to inhibit histone deposition during DNA replication and also disrupt nucleosomal dynamics due to aberrant chromatin condensation. These results also suggest that BE-4-4-4-4 and BE-3-3-3 may produce their cytotoxic effect through slightly different mechanisms.