title: Mycobacterium tuberculosis class II apurinic/apyrimidinic-endonuclease/3'-5' exonuclease III exhibits DNA regulated modes of interaction with the sliding DNA β-clamp.
abstract: The class-II AP-endonuclease (XthA) acts on abasic sites of damaged DNA in bacterial base excision repair. We identified that the sliding DNA β-clamp forms in vivo and in vitro complexes with XthA in Mycobacterium tuberculosis. A novel 239 QLRFPKK245 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 β-clamp located on different domains interact with XthA. The β-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 β-clamp stimulates the activities of XthA primarily by increasing its affinity for the substrate and its processivity. Additionally, loading of the β-clamp onto DNA is required for activity stimulation. A reduction in XthA activity stimulation was observed in the presence of β-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 β-clamp is important for interactions with XthA, while the C-terminal domain predominantly mediates functional interactions in the substrate's presence.
authors.affiliation: Molecular and Structural Biology Division, CSIR-Central Drug Research Institute, Sector 10, Jankipuram Extension, Sitapur Road, Lucknow, Uttar Pradesh, 226031, India.
authors.forename: Taran
authors.initials: T
authors.lastname: Khanam
authors.affiliation: Molecular and Structural Biology Division, CSIR-Central Drug Research Institute, Sector 10, Jankipuram Extension, Sitapur Road, Lucknow, Uttar Pradesh, 226031, India.
authors.forename: Niyati
authors.initials: N
authors.lastname: Rai
authors.affiliation: Molecular and Structural Biology Division, CSIR-Central Drug Research Institute, Sector 10, Jankipuram Extension, Sitapur Road, Lucknow, Uttar Pradesh, 226031, India.
authors.forename: Ravishankar
authors.initials: R
authors.lastname: Ramachandran
mesh_terms: D020816:Amino Acid Motifs; D003198:Computer Simulation; D004249:DNA Damage; D004258:DNA Polymerase III / Q000737:chemistry / Q000378:metabolism*; D004260:DNA Repair; D004269:DNA, Bacterial / Q000378:metabolism*; D043603:DNA-(Apurinic or Apyrimidinic Site) Lyase / Q000737:chemistry* / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000235:genetics; D005090:Exodeoxyribonucleases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D008958:Models, Molecular; D062105:Molecular Docking Simulation; D009154:Mutation; D009169:Mycobacterium tuberculosis / Q000201:enzymology / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D054730:Protein Interaction Domains and Motifs
chemical_list: D004269:DNA, Bacterial; D004268:DNA-Binding Proteins; C101139:beta subunit, DNA polymerase III; D004258:DNA Polymerase III; D005090:Exodeoxyribonucleases; C020133:exodeoxyribonuclease III; D043603:DNA-(Apurinic or Apyrimidinic Site) Lyase