Characterization of Chinese hamster apurinic/apyrimidinic endonuclease (chAPE1)
Abasic sites in DNA are lesions that arise from the hydrolysis of the N-glycosyl bond linking a nitrogenous base to the ribose-phosphate backbone. This base loss occurs spontaneously through DNA damaging agents such as reactive oxygen species, ionizing radiation, and chemotherapeutic agents. Abasic sites can also result from the catalytic activity of a group of DNA glycosylases. It has been estimated that ∼10,000 purines and 200 pyrimidines are lost, spontaneously, in a mammalian cell genome per cell cycle. Thus, abasic sites are potential premutagenic lesions, which can lead to mutations or cell death. Base excision repair pathways have evolved to correct abasic sites and other forms of DNA damage in the cell. Apurinic/apyrimidinic endonuclease is a key enzyme in the base excision repair pathways. AP endonuclease makes a single nick 5′ to the abasic site, leaving a free 3 ′-hydroxyl terminus. This catalytic activity is divalent metal ion dependent. ^ The Chinese hamster apurinic/apyrimidinic endonuclease (chAPE1) gene has been cloned and characterized, however, the protein has not been characterized. To characterize this enzyme and develop a more thorough understanding of the catalytic mechanism of APE1, we over-expressed the cDNA of chAPE1 in sf9 cell line using a baculovirus system. The resulting purified protein was used to characterize the enzymatic activity. ^ Enzymatic kinetic analysis revealed that the kinetic parameters, are similar to those of the human APE1. However, the optimal pH value for its endonuclease activity was established to be 9.5, with suggested significant substrate/product inhibition at pH 7.5, at which human APE1 is believed to have maximum activity. ^ Phosphorylation and dephosphorylation experiments on the recombinant chAPE1 were carried out and the effects of those modifications on the catalytic activity of chAPE1 were also assessed. Casein kinase II inhibits the AP endonuclease activity of chAPE1, while lambda phosphatase appears to be an effective activator of the catalytic activity of this enzyme. Thus, the data of these in vitro experiments suggest that phosphorylation might be an important regulator of the enzymatic activity of chAPE1 in vivo. ^ Evidence for a two-metal ion mechanism for endonuclease catalytic activity was provided by examining the rate of the nicking of abasic DNA as a function of La3+ concentration in the presence of a constant concentration of Mg2+ (at pH 7.5). The effects of various monovalent and divalent cations on the catalytic activity of chAPE1 were also examined. These data indicated that La3+ is an inhibitor of the catalytic activity of chAPE1. The structural modeling of chAPE1 was built, on the basis of our kinetic data and the known crystal structure of human APE, to position the two metal ions in the active site of chAPE1 as well as to interpret the role of the two metal ions in the catalysis. ^
Biology, Molecular|Chemistry, Biochemistry
Man, Dula, "Characterization of Chinese hamster apurinic/apyrimidinic endonuclease (chAPE1)" (2004). ETD Collection for University of Texas, El Paso. AAI3151886.