The HMG1-DNA Interactions at Different Ionic Strengths and in the Presence of cis-Platin Ligands.
It is known that the most abundant non-histone chromatin protein HMG1 exhibit the ability to package DNA into complexes which can penetrate into cells with subsequent expression of the reporter gene. The aim of this work was to study the interaction of the HMG1 with plasmid (pCMV Luci - 6011 bps) and calf thymus DNA. The rat recombinant HMG1 was obtained with an expression system, based on methylotrophic yeast Pichia pastoris. The complexes with input protein/DNA ratio r (w/w) in the range of 0*9 were studied in solutions of different ionic strengths by the methods of analytical ultracentrifugation, circular dichroism (CD) and gel retardation.
The results obtained, have suggested, that HMG1 preferentially binds to the supercoiled DNA and the manner of interaction changes in the vicinity of r=5. It has been confirmed by CD analysis that direct protein-DNA interaction has occurred at the values of r<4. We have estimated HMG1 binding site on the DNA at this mode of interaction as ~15 bp. At further increase of protein concentration the protein-protein interactions were involved in the formation of complex. The dependence of complex formation on salt concentration has been investigated up to the 1M NaCl. It has been shown that the complexes obtained at high protein content were stable in solutions with extremely high ionic strength. It was also shown by CD measurements that similar changes of local DNA conformaton have been induced by the interaction with HMG1 and platinum ligand (cis-DDP). A direct interaction between HMG1 and cisplatin adduct has been observed.
Alexander Polyanichko1, Svetlana Davydenko2, Elena Chikhirzhina and Vladimir Vorob'ev2
1Saint-Petersburg State University;