Mechanistic decoupling of exonuclease III multifunctionality into AP endonuclease and exonuclease activities at the single-residue level

Bacterial exonuclease III (ExoIII) is a multifunctional enzyme that uses a single active site to perform two conspicuous activities: (i) apurinic/apyrimidinic (AP)-endonuclease and (ii) 3 '-> 5 ' exonuclease activities. The AP endonuclease activity results in AP site incision, while the exonuclease activity results in the continuous excision of 3 ' terminal nucleobases to generate a partial duplex for recruiting the downstream DNA polymerase during the base excision repair process (BER). The key determinants of functional selection between the two activities are poorly understood. Here, we use a series of mutational analyses and single-molecule imaging to unravel the pivotal rules governing these endo- and exonuclease activities at the single amino acid level. An aromatic residue, either W212 or F213, recognizes AP sites to allow for the AP endonuclease activity, and the F213 residue also participates in the stabilization of the melted state of the 3 ' terminal nucleobases, leading to the catalytically competent state that activates the 3 '-> 5 ' exonuclease activity. During exonucleolytic cleavage, the DNA substrate must be maintained as a B-form helix through a series of phosphate-stabilizing residues (R90, Y109, K121 and N153). Our work decouples the AP endonuclease and exonuclease activities of ExoIII and provides insights into how this multifunctional enzyme controls each function at the amino acid level.

Automated summary

Bacterial exonuclease III (ExoIII) is a multifunctional enzyme that has both apurinic/apyrimidinic (AP)-endonuclease and 3'->5' exonuclease activities. Through mutational analysis and single-molecule imaging, researchers have discovered the key amino acid residues that determine the functional selection between these two activities. Additionally, they found that stabilizing the melted state of the 3' terminal nucleobases is crucial for activating the exonuclease activity. Maintaining the DNA substrate as a B-form helix during the exonucleolytic cleavage process is also essential. This study provides insights into the mechanism of ExoIII's dual functionalities at the amino acid level.