A comparative study of recombinant mouse and human apurinic/apyrimidinic endonuclease
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Mammalian apurinic/apyrimidinic endonuclease (APE1) initiates the repair of abasic sites (AP-sites), which are highly toxic, mutagenic, and implicated in carcinogenesis. Also, reducing the activity of APE1 protein in cancer cells and tumors sensitizes mammalian tumor cells to a variety of laboratory and clinical chemotherapeutic agents. In general, mouse models are used in studies of basic mechanisms of carcinogenesis, as well as pre-clinical studies before transitioning into humans. Human APE1 (hAPE1) has previously been cloned, expressed, and extensively characterized. However, the knowledge regarding the characterization of mouse APE1 (mAPE1) is very limited. Here we have expressed and purified full-length hAPE1 and mAPE1 in and from E. coli to near homogeneity. mAPE1 showed comparable fast reaction kinetics to its human counterpart. Steady-state enzyme kinetics showed an apparent K m of 91 nM and k cat of 4.2 s−1 of mAPE1 for the THF cleavage reaction. For hAPE1 apparent K m and k cat were 82 nM and 3.2 s−1, respectively, under similar reaction conditions. However, k cat/K m were in similar range for both APE1s. The optimum pH was in the range of 7.5–8 for both APE1s and had an optimal activity at 50–100 mM KCl, and they showed Mg2+ dependence and abrogation of activity at high salt. Circular dichroism spectroscopy revealed that increasing the Mg2+ concentration altered the ratio of “turns” to “β-strands” for both proteins, and this change may be associated with the conformational changes required to achieve an active state. Overall, compared to hAPE1, mAPE1 has higher K m and k cat values. However, overall results from this study suggest that human and mouse APE1s have mostly similar biochemical and biophysical properties. Thus, the conclusions of mouse studies to elucidate APE1 biology and its role in carcinogenesis may be extrapolated to apply to human biology. This includes the development and validation of effective APE1 inhibitors as chemosensitizers in clinical studies.
KeywordsAPE1 Base excision repair CD
Base excision repair
Mouse apurinic/apyrimidinic endonuclease 1
Human apurinic/apyrimidinic endonuclease 1
The authors thank Prof. Sankar Mitra of UTMB, Galveston, Texas for providing the mAPE1 cDNA. The authors thank Dr. Amrita Cheema for proteomics experiments performed at the Proteomics and Metabolomics Shared Resource of the Lombardi Comprehensive Cancer Center. The authors also thank Mrs. Jordan Woodrick for critically reading the paper. The study was supported by NIH grants RO1 CA 92306 (RR), RO1 CA 113447 (RR) and ACS grant ACS-IRG-92-152-17 (SA).
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