Sequence Specificity Of Copper Zinc Superoxide Dismutase Binding To DNA

In microorganisms,regulatory metalloproteins regulate gene expression in response to environmental reactive oxygen species(ROS)changes.There might also be similar metalloprotein-based regulatory mechanism in mammalian.As an antioxidant metalloenzyme maintaining intracellular ROS homeostasis,copper-zinc superoxide dismutase(SOD1)widespreadly exists in different kinds of cells.The increased concentration of H2O2 makes SOD1 phosphorylate and translocate to nucleus to regulate gene expression in yeast cells.Hence,we proposed that SOD1 might be a gene expression regulatory protein that responses intracellular ROS levels.In order to confirm this hypothesis,immunofluorescence assay performed by Xiang Li indicated that the increased levels of H2O2 induced SOD1 translocation to the nucleus in HeLa cells.DNA sequences that interact with SOD1 were acquired by chromatin precipitation-sequencing assay.This work confirmed the affinity between SOD1 and multiple DNA in solution,researched the effects of various condition on SOD1-DNA binding,and then identified the binding sites in high preference DNA sequence.The main results are as follows:1.This work confirmed that SOD1 is a DNA binding protein.The affinity ability of WT-SOD1 with different DNA sequences was determined,and the S1 was the highest preference sequence,double stranded DNA with 12 bases(ATGGAATGGAAT).2.The affinity of WT-SOD1 to S1 sequence is higher than its mutants A4V and H46R-H48Q.3.Under different pH conditions,the combination of WT-SOD1 and H46R-H48Q with S1 was not obviously affected.4.Under oxidative condition,the combination of WT-SOD1 and A4V with SI was weakened,and the combination of H46R-H48Q with S1 was enhanced.Under reduced condition,the combination of WT-SOD1 and S1 was slightly enhanced,the combination of WT-SOD1 and A4V with S1 was not obviously unaffected.5.SOD1 and mutants binding with 31 sequence was weakened by Cu2+ or Zn2+chelator.The metal ions of SOD1 affected its combination with DNA by stabilizing the protein structure.6.SOD 1 binding sites of SL(a derivative of S1)is GGA.The study of SOD 1-DNA binding provided an evidence that SOD1 might be a gene expression regulatory protein responsing intracellular ROS changes in mammalian cells.Identification of SOD1 binding sites also established foundation to study the structure of SOD1-DNA complexes and understand the molecular mechanism of SOD1 as a regulatory metallprotein.