0428同步年報-2021-全

Life Science 053 Fig. 1 : (a,b) APE1-recessed dsDNA product complex structures and conformational changes of the RM bridge. (c) The RM bridge occupies the space of the active site and separates the active site into the catalytic site and a product pocket. (d) A magnified view of the active site when APE1 works as an endonuclease or exonuclease. The structural comparison reveals the structural basis of the base preference of APE1. [Reproduced from Ref. 1 and 2] APE1 Follows the Induced Space-Filling Mechanism to Distinguish and Processing Various DNA Substrates Combining APE1-DNA complex structures and biochemical assays, we established a space-filling model to depict how APE1 distinguishes various substrates and to reveal the structural basis of the drug-resistant mechanism associated with APE1. A PE1 is a DNA-repair enzyme with both endonuclease and exonuclease activity. As an endonuclease, the apurinic/ apyrimidinic site (AP site)-specific cleavage of APE1 is an essential process to repair oxidized nucleotides in the base excision repair (BER) path. As an exonuclease, APE1 digests various deoxynucleotides at the 3’- termini of matched or 1 nt-mismatched duplex DNA without base preference. The non-specific exonuclease activity of APE1 leads to nucleoside analogue-typed anti-cancer drug resistance. Excision of the 3’-end-matched base pair by APE1 in various structural duplex DNA, such as gapped, nicked and recessed dsDNA, is vital for a series of DNA processing paths, including nucleotide incision repair, Trinucleotide repeat expansion-related BER, single-strand breaks and apoptosis. Unlike the well studied endonuclease activity, the lack of structural information leaves the molecular details of the base and structural preference of APE1 exonuclease activity still missing. In this study, we determined two terminal-binding structures of the APE1-dsDNA complex and demonstrated an induced space-filling model to reveal the structural basis of its exonuclease activity. The X-ray diffraction data sets were collected at beamlines TLS 13B1 , TLS 15A1 , and TPS 05A of the NSRRC. 1 After DNA binding, Arg176 and Met269 of APE1 are induced to form a bridge-like structure (RM bridge) occupying the active site and creating a narrow channel ( Figs. 1(a-c) ). A RM bridge separates the active site into the catalytic site and a product pocket in which the unique structure conducts the different substrate specificity of APE1 endo- and exo-nuclease activity. In the endonuclease manner, APE1 is an AP site-specific nuclease because the steric hindrance from the downstream nucleotide and narrow product pocket makes a normal nucleotide that cannot be accommodated ( Fig. 1(d) ). In the