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2019 Biophysical Society Meeting, Baltimore Maryland

Metal ion interactions are significant aspects of protein-nucleic acid binding and the enzymatic activity associated with these proteins. Metal ion dependence for structure and function of protein-nucleic acid complexes are well-studied, but in many cases, the exact stoichiometry of metal ion release or absorption has been experimentally challenging to determine. A particular case is that of the bacterial DNA repair enzyme, RecBCD, which has a magnesium ion (Mg2+) dependent base-pair disruption activity key to initiation of its helicase activity. In this project, we propose to utilize the Mg2+ binding fluorescent dye, 8-hydroxyquinoline-5-sulfonic acid (HQS), to calculate the amount of Mg2+ that is removed from solution by uptake of the RecBCD-DNA binding. These results indicate that an HQS fluorescent assay is sensitive to [Mg2+] changes involved with ATP:Mg2+ binding, DNA:Mg2 interactions, and the RecBCD:DNA:Mg2 complex.