EFFECTS OF SPERMINE AND ITS CYTOTOXIC ANALOGS ON NUCLEOSOME FORMATIONON TOPOLOGICALLY STRESSED DNA IN-VITRO

We investigated the effects of the polyamine spermine and two of its c ytotoxic 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 for mation of nucleosomes on negatively and positively supercoiled DNA in vitro. Histones H2A, H2B, H3 and H4 were reconstituted onto DNA to for m nucleosomes and the polyamines were added either before or after his tone 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 deg rees, high concentrations of topoisomerase I were able to relax the ag gregated DNA and the helical pitch was found to be unaltered in the ag gregates. When histones were associated with negatively coiled DNA, th e polyamine-induced aggregation did not alter nucleosome structure. Th e 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 l east effective. These analogs were also extremely effective in inhibit ing histone deposition onto DNA. A potential mechanism for the action of these analogs is both to inhibit histone deposition during DNA repl ication and also disrupt nucleosomal dynamics due to aberrant chromati n condensation. These results also suggest that BE-4-4-4-4 and BE-3-3- 3 may produce their cytotoxic effect through slightly different mechan isms.