Chinese hamster apurinic/apyrimidinic endonuclease (chAPE1) expressed in sf9 cells reveals that its endonuclease activity is regulated by phosphorylation

Abstract

Apurinic/apyrimidinic endonuclease (APE), an essential DNA repair enzyme, initiates the base excision repair pathway by creating a nick 5′ to an abasic site in double‐stranded DNA. Although the Chinese hamster ovary cells remain an important model for DNA repair studies, the Chinese hamster APE (chAPE1) has not been studied in vitro in respect to its kinetic characteristics. Here we report the results of a kinetic study performed on cloned and overexpressed enzyme in sf9 cells. The kinetic parameters were fully compatible with the broad range of kinetic parameters reported for the human enzyme. However, the activity measures depended on the time point of the culture. We applied inductivity coupled plasma spectrometry to measure the phosphorylation level of chAPE1. Our data showed that a higher phosphorylation of chAPE1 in the expression host was correlated to a lower endonuclease activity. The phosphorylation of a higher activity batch of chAPE1 by casein kinase II decreased the endonuclease activity, and the dephosphorylation of chAPE1 by lambda phosphatase increased the endonuclease activity. The exonuclease activity of chAPE1 was not observed in our kinetic analysis. The results suggest that noticeable divergence in reported activity levels for the human APE1 endonuclease might be caused by unaccounted phosphorylation. Our data also demonstrate that only selected kinases and phosphatases exert regulatory effects on chAPE1 endonuclease activity, suggesting further that this regulatory mechanism may function in vivo to turn on and off the function of this important enzyme in different organisms.

Chinese hamster AP endonuclease operates by a two‐metal ion assisted catalytic mechanism

“…chAPE1 was expressed and purified as described [16]. For more details please refer to the Supplementary data.…”

“…The kinetic measurements were carried out according to the protocols as published [16]. For more details please refer to [16] and the Supplementary data, especially Figs. S1 and S2.…”

“…The tetrahydrofuran (F) containing synthetic DNA is an abasic DNA substrate analog widely used in studies of human APE1 [10]. An example of assay results with a duplex DNA containing tetrahydrofuran as a substrate can be found in [16]. The effect of titrating La 3+ on AP endonuclease activity was investigated in a reaction mix containing 5 mM Mg 2+ .…”

“…The Chinese hamster AP endonuclease (chAPE1) gene (92% similarity with human APE1 and 94% with both mouse APEX and rat APEN) was cloned and characterized [15]. The cDNA of chAPE1 was subcloned into baculovirus vector and the recombinant protein was expressed in sf9 cell line and its endonuclease activity characterized [16]. The chAPE1 has a endonuclease activity level comparable to the human counterpart APE1 and its endonuclease activity is controlled by phosphorylation [16].…”

“…The cDNA of chAPE1 was subcloned into baculovirus vector and the recombinant protein was expressed in sf9 cell line and its endonuclease activity characterized [16]. The chAPE1 has a endonuclease activity level comparable to the human counterpart APE1 and its endonuclease activity is controlled by phosphorylation [16].…”