Investigating the effects of mutated radical holding residues on DNA charge transport
DNA primase is essential in the synthesis of new DNA during replication. It is important because no other replicative polymerases can initiate the synthesis of a DNA strand without an initial primer. Primase contains a redox active 4Fe-4S cluster in its DNA binding domain, p58C. We have found that this cofactor can engage in DNA charge transport. In DNA charge transport, electrons are passed through an intact DNA duplex between redox centers. In primase, charge transport is mediated by a pathway of residues between the DNA binding site and the 4Fe-4S cluster. Mutating these residues can abrogate DNA charge transport. We hypothesize that DNA charge transport is important for primase function. To investigate this, I have generated and biophysically characterized three different variants of yeast p58C. Yeast was chosen because it is similar to the human protein and is easier to manipulate genetically. The mutations were made through site directed mutagenesis. We assessed structural integrity and thermal stability using x-ray crystallography and circular dichroism. We also probed DNA binding ability of the mutants by performing fluorescence-based DNA binding assays. These variants will be sent to our collaborators at CalTech for electrochemical analysis. If these variants are charge transport deficient, they will be incorporated into the yeast genome to assess how charge transport deficiency affects DNA replication in the cell. These experiments will shed more light on the role of DNA charge transport in primase activity and DNA replication.