Comparative Analysis of the 3′–5′ Exonuclease Activity of Human APE1 and Arabidopsis ARP AP Endonucleases

• A long N-terminal region with a SAP domain modulates the AP endonuclease and 3′–5′ exonuclease activities of ARP. • Both APE1 and ARP exhibit limited processivity but differ in their efficiency at processing DNA substrates of different lengths. • Both enzymes remove matched and mismatched nucleotides with similar efficiency, unaffected by the identity of the opposing base. • Two DNA intercalating residues (R177/M270 in APE1; R488/D395 in ARP) influence substrate specificity and exonucleolytic cleavage. AP endonucleases are essential for repairing abasic (apurinic/apyrimidinic, AP) sites in DNA, which arise from spontaneous base loss or enzymatic base excision. The main AP endonucleases in mammals and plants are APE1 and ARP, respectively, two orthologous enzymes that belong to the ExoIII family. Members of this family play multifunctional roles in DNA repair and combine AP endonuclease activity with additional functions such as 3′–5′ exonuclease activity. APE1 and ARP exhibit structural differences that influence their AP endonuclease activity, but the molecular features underlying differences in exonuclease activity remain unknown. Here, we systematically compare APE1 and ARP to elucidate the structural and mechanistic basis of their exonuclease function. We found that in ARP an extended N-terminal region with a SAP domain, absent in APE1, modulates its AP endonuclease and 3′–5′ exonuclease activities. Both APE1 and ARP exhibit low processivity, but they differ in their efficiency when processing DNA substrates of varying lengths. While both enzymes remove matched and mismatched nucleotides with equal efficiency and show no preference for the opposite base, their activity is modulated by two DNA-intercalating residues (R177/M270 in APE1; R488/D395 in ARP), which influence specificity and cleavage patterns. These findings reveal mechanistic and structural differences that may reflect the functional specialization and evolutionary divergence of plant and mammalian AP endonucleases.