Mycobacterium tuberculosis class II apurinic/apyrimidinic-endonuclease/3′-5′ exonuclease III exhibits DNA regulated modes of interaction with the sliding DNA β-clamp

The class-II AP-endonuclease (XthA) acts on abasic sites of damaged DNA in bacterial base excision repair. We identified that the sliding DNA beta-clamp forms in vivo and in vitro complexes with XthA in Mycobacterium tuberculosis. A novel (239)QLRFPKK(245) motif in the DNA-binding domain of XthA was found to be important for the interactions. Likewise, the peptide binding-groove (PBG) and the C-terminal of beta-clamp located on different domains interact with XthA. The beta-clamp-XthA complex can be disrupted by clamp binding peptides and also by a specific bacterial clamp inhibitor that binds at the PBG. We also identified that beta-clamp stimulates the activities of XthA primarily by increasing its affinity for the substrate and its processivity. Additionally, loading of the beta-clamp onto DNA is required for activity stimulation. A reduction in XthA activity stimulation was observed in the presence of beta-clamp binding peptides supporting that direct interactions between the proteins are necessary to cause stimulation. Finally, we found that in the absence of DNA, the PBG located on the second domain of the beta-clamp is important for interactions with XthA, while the C-terminal domain predominantly mediates functional interactions in the substrate's presence.