Nucleolytic Activity And DNA-Accelerated Aggregation Of Copper, Zinc Superoxide Dismutase

Amyotrophic lateral sclerosis (ALS) is one of the most common adult neurodegenerative diseases with unknown causes. Although it is well established that SOD1 mutants involve development of ALS, and cause motor neuron death through an as-yet unidentified gain of one or more injurious properties, little is currently known about the roles of SOD1 in neurocytotoxicity. DNA is the source of genetic information and possesses many important roles in life process. Even the weak damage in DNA could have profound effect on viability and genetic stability. Moreover, DNA damage in human cells and organs has been found to be associated with neurodegenerative disease. Here, the research on the interaction between SOD1 and DNA has been carried out. The main results are as follows:1. A new activity that the SOD1 and its apo form possess a divalent metal-dependent nucleolytic activity was confirmed by UV-visible absorption titration of calf thymus DNA (ctDNA) with the SOD1, fluorescence quenching of SOD1 by ctDNA, and by gel electrophoresis monitoring conversion of DNA from the supercoiled DNA to nicked and linear forms, and fragmentation of a linearλDNA. Moreover, the DNA cleavage activity was examined in detail under certain reaction conditions. The steady state study indicates that DNA cleavage supported by both forms of SOD1 obeys Michaelis-Menten kinetics. On the other hand, the assays with some other proteins indicate that this new gain function is specific to some proteins including the SOD1. Therefore, this study reveals that the divalent metal-dependent DNA cleavage activity is an intrinsic property of SOD1, which is independent of its natural metal sites.2. The roles of exogenous divalent metals in the nucleolytic activity were explored in detail by a series of biochemical experiments. Based on a non-equivalent multi-site binding model, affinity of a divalent metal for the enzyme-DNA complex was determined by absorption titration, indicating that the complex can provide at least a high and a low affinity site for the metal ion. These mean that the SOD1 may use a''two exogenous metal ion pathway''as a mechanism in which both metal ions are directly involved in the catalytic process of DNA cleavage. In addition, the pH versus DNA cleavage rate profiles can be fitted to two ionizing group models, indicating the presence of a general acid and a general base in catalysis. A model that requires histidine residues, metal bound water molecules and two hydrated metal ions to operate in concert could be used to interpret the catalysis of DNA hydrolysis, supported by the dependences of loss of the nucleolytic activity on time and on the concentration of the specific chemical modifier to the histidine residues on the enzyme.3. The aggregation behavior of SOD1 in the presence of DNA were examined under acidic conditions, which could mimic the effect of mutations and reflect the practical process done under physiological conditions to a high extent. Several forms of double-stranded DNA were tested to trigger the SOD1 aggregation by light scattering, single- and double-fluorescence imaging with the dyes, atomic force microscopy, and direct observations under visible light. The results reveal that DNA acts as a template for accelerating the formation of SOD1 aggregates and is incorporated into SOD1 aggregates. A significant alteration in hydrophobicity of SOD1 caused by both low pH and interactions with DNA, and the enrichment in SOD1 along DNA double strands are two main reasons responsible for DNA-accelerated SOD1 aggregation.4. The morphology of DNA-accelerated SOD1 aggregates was examined by transmission electron microscope, atomic force microscopy and fluorescence imaging of ThS. Several types of SOD1 aggregates were observed, which depend on the concentration of SOD1 and DNA, and the type of DNA molecules. DNA formed a compact structure in the high ratio of DNA to SOD1, and an incompact structure in the low ratio. The results reveal that both SOD1 aggregation and DNA condensation are coupled each other. Some DNA condensates could aggregate together to form fractal structures on mica and class, which followed the diffusion limited aggregation model.5. The acceleration effect of SOD1 aggregation in vitro upon addition of single-stranded DNA (ssDNA) of 24 nucleotides was examined under acidic conditions. ssDNA was tested to trigger the SOD1 aggregation by light scattering, single- and double-fluorescence imaging with the dyes, direct observations under visible light,atomic force microscopy and transmission electron microscope. The results reveal that ssDNA can accelerate the formation of ssDNA-SOD1 aggregate monomer, oligomeric aggregate, microaggregate, and macroaggregate. A significant alteration in hydrophobicity of SOD1 caused by both low pH and interactions with ssDNA, removal of the positive net charges of SOD1 by ssDNA, and the enrichment in SOD1 along ssDNA are driving forces for the rapid SOD1 aggregation. All these results indicate that the ssDNA-accelerated formation of insoluble SOD1 aggregates can act as a potential pathway to avoid accumulation of soluble SOD1 oligomeric intermediates. The small DNAs that are easily synthesized to target at protein oligomers might lower pathological consequences of SOD1.6. The acceleration effect of oxidized SOD1 aggregation in vitro upon addition of DNA was examined by gel electrophoresis, light scattering, and transmission electron microscope under physiological conditions. The results reveal that DNA can accelerate SOD1 aggregation when both SOD1 and DNA are in oxidative solution. A significant increase in hydrophobicity of SOD1 caused by oxidative modification, and the enrichment in SOD1 along DNA double strands are two main reasons responsible for the accelerated SOD1 aggregation. This result also implies that SOD1 of any one form with increased hydrophobicity, whatever caused by mutation or exogenous factor, may be accelerated to form aggregates due to the enrichment effect of DNA. This abnormal interaction with DNA may be a toxic gain of function of misfolded SOD1, which could cause ALS.