Endonuclease G 切割核酸機制之研究

Abstract

Endonuclease G (EndoG) is an apoptotic nuclease involved in nucleosomal fragmentation of DNA. Here we expressed and purified the human EndoG in Escherichia coli with the use of pET expression vector. Recombinant human EndoG initiated single strand breaks in plasmids, and the accumulation of nicks resulted in the double strand breaks of DNA. In this study, histidine residues involved in the catalytic activity of human EndoG have been characterized. Diethyl pyrocarbonate (DEPC) modification and hydroxylamine restoration assay indicated that histidine residues were responsible for the catalysis of human EndoG. A survey of the ββα-Me finger and H-N-H nuclease, it was noticed that a good structural superposition in the H-N-H region could be achieved for NucA (1ZM8), I-PpoI (1A73), Colicin E9 (1XBI), m5C-specific REase McrA, and T4 endonuclase VII (1EN7). The site-directed mutants of H-N-H motif of EndoG were further constructed and analyzed by kinetic studies. Kinetic analysis indicated that replacement of histidine-141, aspasragine-163, and aspasragine-172 with alanine residue, resulted in a drastic reduction on catalytic efficiency (Kcat/Km). These results suggested that H141, N163 and N172 were critical for catalysis. The enzyme activity of wild-type EndoG was inhibited at higher magnesium concentration. EndoG mutants shared similar profiles with wild-type EndoG. However, H141A and E271A achieved the maximal activity at a 60-fold higher magnesium concentration than wild-type EndoG. These findings suggested that magnesium ion might be coordinated by His141 and E271A of human EndoG. Because EndoG has been proved to exhibit the a sequence-specific cleavage activity, we further analyzed the EndoG mutants of H141A, N163A, and N172A in the a sequence cleavage. These results indicated that H141, N163 and N172 were critical for substrate specificity. Taken together, our data suggested that the H-N-H motif is present in human EndoG, the domain H-N-H of human EndoG performed magnesium binding and specificity of cutting function.