title: Characterization of E. coli manganese superoxide dismutase binding to RNA and DNA.
abstract: Bacterial manganese superoxide dismutase (MnSOD) has been shown to localize to the chromosomal portion of the cell and impart protection from ionizing radiation to DNA. The binding affinity of bacterial MnSOD to non-sequence specific double stranded oligomeric DNA has been quantitated previously by nitrocellulose filter binding and gel shift assays. In the current study we have examined the equilibrium binding of Escherichia coli MnSOD to poly(U), poly(A), poly(C), poly(dU) and double-stranded (ds) DNA. Equilibrium association constant, Kobs, was measured by monitoring intrinsic tryptophan fluorescence quenching. Based on the extent of quenching, Kobs was determined as a function of monovalent salt (MX) concentration and type, as well as temperature, from which ΔG°obs and ΔH°obs were determined. It was found that the polynucleotides bind to MnSOD in the following affinity hierarchy, poly(dU)>poly(U)>dsDNA>poly(A)>poly(C). The differences in the hierarchy were not large in magnitude as the poly(dU) bound with less than a 100-fold higher affinity than poly(C) at any given [MX]. For each polynucleotide, Kobs decreases only slightly with increasing [K(+)], surprising for a relatively non-specific nucleic acid protein. Thus, our finding that MnSOD can bind to RNA leads to the possibility that MnSOD may confer protection to RNA, as well. This is, as of yet, untested. Typically one would expect strong electrostatic interactions to dominate a non-specific binding event like that, but our results show an unexpectedly strong non-electrostatic contribution to the binding.
authors.affiliation: Department of Chemistry, John Carroll University, 20700 North Park Boulevard, University Heights, OH 44118, USA.
authors.forename: Angela C
authors.initials: AC
authors.lastname: Smolik
authors.affiliation: Department of Chemistry, John Carroll University, 20700 North Park Boulevard, University Heights, OH 44118, USA.
authors.forename: Lana
authors.initials: L
authors.lastname: Bengez-Pudja
authors.affiliation: Department of Chemistry, John Carroll University, 20700 North Park Boulevard, University Heights, OH 44118, USA.
authors.forename: Iteen
authors.initials: I
authors.lastname: Cheng
authors.affiliation: Department of Chemistry, John Carroll University, 20700 North Park Boulevard, University Heights, OH 44118, USA. Electronic address: dmascotti@jcu.edu.
authors.forename: David P
authors.initials: DP
authors.lastname: Mascotti
keywords: Fluorescence; Manganese superoxide dismutase (MnSOD); Polynucleotide; Protein–DNA interaction; Protein–RNA interaction; Thermodynamics