RESUMO
M-DNA is a novel duplex conformation in which metal ions such as Co2+, Ni2+ or Zn2+ replace the imino protons of every base pair. An ethidium fluorescence assay was used to estimate lesions in M-DNA induced by gamma- and UV radiation. General damage to DNA was assessed from the loss of ethidium fluorescence after irradiation of calf thymus DNA. Crosslinks were measured from the return of ethidium fluorescence after a heating and cooling step. Strand breaks were estimated from the loss of fluorescence in covalently closed circular plasmid DNA after a heating and cooling step. For the Co2+ form of M-DNA, gamma-radiation caused the very efficient formation of crosslinks which was not observed with B-DNA nor with the Ni2+ or Zn2+ forms of M-DNA. The crosslinks occurred in both A-T and G-C base pairs but did not form in the presence of a free radical scavenger. Crosslinks induced by UV radiation also formed at a faster rate in the Co2+, Ni2+ and Zn2+ forms of M-DNA compared to B-DNA; crosslinking occurred in all DNA but was more prominent in AT-rich sequences and was not inhibited by a free radical scavenger. Therefore, the presence of certain metal ions may lead to large increases in the formation of radiation-induced crosslinks in DNA.
Assuntos
DNA/efeitos da radiação , Reagentes de Ligações Cruzadas , DNA/química , DNA/efeitos dos fármacos , Dano ao DNA , Raios gama , Concentração de Íons de Hidrogênio , Técnicas In Vitro , Metais/farmacologia , Conformação de Ácido Nucleico/efeitos dos fármacos , Conformação de Ácido Nucleico/efeitos da radiação , Fotoquímica , Espectrometria de Fluorescência , Raios UltravioletaRESUMO
M-DNA is a complex of DNA with divalent metal ions (Zn(2+), Co(2+), or Ni(2+)) which forms at pH conditions above 8. Upon addition of these metal ions to B-DNA at pH 8.5, the pH decreases such that one proton is released per base-pair per metal ion. Together with previous NMR data, this result demonstrated that the imino proton in each base-pair of the duplex was substituted by a metal ion and that M-DNA might possess unusual conductive properties. Duplexes of 20 base-pairs were constructed with fluorescein (donor) at one end and rhodamine (acceptor) at the other. Upon formation of M-DNA (with Zn(2+)) the fluorescence of the donor was 95 % quenched. Fluorescence lifetime measurements showed the presence of a very fast component in the decay kinetics with tau=10 ps. The fast component was absent in B-DNA and in M-DNA lacking an acceptor chromophore; a result which is only consistent with electron transfer. Efficient signal transduction was also observed between the two fluorophores separated by 54 base-pairs (over 150 A) in an M-DNA duplex. The addition of a sequence-specific DNA-binding protein prevented the flow of electrons and this was reversed by protease digestion. Therefore, M-DNA behaves as a molecular wire and could be manipulated to prepare self-assembling electronic circuits.