A study on the activity change of hAPE1 mutation using smFRET

Abstract

Apurinic/apyrimidinic endonuclease 1 (APE1) is an essential protein in BER that creates nick in the 5' portion of the AP site. This hAPE1 has a similar structure to ExoIII of E. coli. However, despite these structural similarities, hAPE1 has only a very weak nucleic acid exonuclease activity unlike ExoIII. In order to understand the reason for the difference between hAPE1 and ExoⅢ and to further understand the function of the structure according to the sequence of APE1, a hAPE1 mutation study was conducted. The mutation of hAPE1 was conducted in the direction of increasing the exonuclease activity, which was conducted by Donghun Lee, a researcher at our laboratory. We conducted an experiment using smFRET to find out how this APE1 mutation increased the exonuclease activity. We used the hAPE1 v6 (D70N, E126K, F266A, W280A, M270A, N229K) mutant, which has the best exonuclease activity, to find out whether the protein's exonuclease activity is processive. hAPE1 v6 mutant appeared to be a processive exonuclease in experiments with smFRET. In addition, when compared with non-processive Exo III, it was shown that hAPE1 v6 mutant was a processive exonuclease. Other hAPE1 mutants were also tested in the same way, and hAPE1 v4 (D70N, E126K, F266A, W280A) and v5 (D70N, E126K, F266A, W280A, M270A) mutants showed a slightly processive exonuclease, although lower than v6. These hAPE1 mutants were analyzed in-silico, and based on this, a model was presented on how N229, F266, M270, and W280 of hAPE1 affect the function of processivity of exonuclease activity. These results would reveal the key principle to understanding the relationship between function and residue inAPE1 active site.