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Mycobacterium tuberculosis class II apurinic/apyrimidinic‐endonuclease/3′‐5′ exonuclease III exhibits DNA regulated modes of interaction with the sliding DNA β‐clamp
Author(s) -
Khanam Taran,
Rai Niyati,
Ramachandran Ravishankar
Publication year - 2015
Publication title -
molecular microbiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.857
H-Index - 247
eISSN - 1365-2958
pISSN - 0950-382X
DOI - 10.1111/mmi.13102
Subject(s) - biology , dna clamp , processivity , ap site , exonuclease , dna , ap endonuclease , biophysics , endonuclease , exonuclease iii , base excision repair , microbiology and biotechnology , biochemistry , dna polymerase , dna repair , gene , reverse transcriptase , rna , escherichia coli
Summary 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 M ycobacterium 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 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.