0428同步年報-2021-全

054 ACTIVITY REPORT 2021 exonuclease manner, the last nucleotide base can rotate and be placed into another region of the product pocket ( Fig. 1(d) ). In addition, the product pocket is composed of hydrophobic residues that cannot specifically interact with nitrogenous bases with hydrogen bonds. These two structural features make APE1 exonuclease without a base preference. This induced space-filling mechanism also clarifies the selection mechanism for the structural preference of APE1 exonuclease on dsDNA that is critical to identify the role of APE1 on various DNA repair paths. First, APE1 prefers to digest dsDNA with blunt-end and 1 nt-mismatch rather than the 2 nt-mismatch ( Fig. 2(a) ), because the narrow product pocket with steric hindrance cannot accommodate two nucleotides; APE1 can thus difficultly digest substrates with more than 2 nt- mismatched bases ( Fig. 2(b) ). In contrast, the nicked dsDNA with flank 5’-phosphate is an unfavourable substrate for the APE1 exonucleolytic cleavage ( Fig. 2(c) ). The steric hindrance between the base region of 3’-terminal deoxyribonucleotide and the downstream 5’-phosphate group leads to an unfavourable cleavage of 5’-phosphate nicked dsDNA ( Fig. 2(d) ). This hypothesis is proved with nuclease activity assays in vitro. With the substrates without steric hindrance, such as nicked dsDNA with the flank 5’-hydroxy group and gapped dsDNA, the exonuclease activity of APE1 is no longer restricted ( Figs. 2(c) and 2(e) ). In summary, the DNA binding-induced RM bridge fills the space of the APE1 active site to create a narrow channel-liked structure. The narrow hydrophobic product pocket enables the APE1 with different base selection mechanisms in endo- and exo-nuclease activity. When APE1 works as an endonuclease, it targets only the AP site in the middle of dsDNA. When APE1 works as an exonuclease, it removes any nucleotide at the 3’-terminal of duplex DNA without preference. Additionally, the steric hindrance of the product pocket weeds out the dsDNA with longer 3’-overhang and nicked dsDNA with flank 5’-phosphate to construct the structural preference of APE1. Our unprecedented induced space-filling model clarifies the base and structural selection mechanisms of APE1 in processing dsDNA and paves the way to understand the cellular functions and drug resistance associated with APE1. 1,2 (Reported by Tung-Chang Liu and Jung-Yu Liu, , National Yang Ming Chiao Tung University) Fig. 2 : (a) Exonuclease activity of mAPE1 in digesting dsDNA with blunt-end, 1-nt mismatch and 2-nt mismatch. dsDNA with 2-nt mismatch is a poor substrate of APE1 exonuclease. (b) An induced space-filling model depicts why dsDNA with 2-nt mismatch is a poor substrate. (c) Exonuclease activity of mAPE1 in digesting nicked dsDNA with or without flank 5’-phosphate. (d) The induced space-filling model depicts how the APE1 exonuclease activity is blocked by nicked dsDNA with flank 5’-phosphate, and why APE1 can digest gapped dsDNA. (e) Nuclease activity of APE1 on gapped dsDNA. [Reproduced from Ref. 1 and 2]