Regulation of futile ligation during early steps of BER in M. tuberculosis is carried out by a ?-clamp-XthA-LigA tri-component complex

The Class-II AP-endonuclease (XthA) is a mycobacterial DNA base excision repair (BER) pathway enzyme that functions in the initial steps. It acts on DNA substrates that contain abasic sites to create nicks with 3 '-hydroxyl (OH) and 5 '-deoxyribose phosphate (5 '-dRP) moieties. The NAD+-dependent DNA ligase (LigA) is the terminal player in mycobacterial BER and seals such nicks efficiently. Here, we demonstrate that the Mtb beta-clampMtbXthA complex that exists in the initial steps of BER engages with MtbLigA to form a novel tri-component BER complex. Size exclusion chromatography (SEC) experiments analysis show that the three proteins interact with equimolar stoichiometry. Small angle X-ray scattering (SAXS) analysis and associated studies reveal that the apo tri-component BER-complex adopts an extended conformation where MtbXthA is sandwiched between the Mtb beta clamp and MtbLigA. The studies support that in the apo-complex MtbXthA binds subsite-I of Mtb beta-clamp through 239QLRFPKK245 motif and to MtbLigA by 104DGQPSWSGKP113 motif simultaneously. However, the complex adopts a less-extended conformation in the presence of substrate DNA, where MtbXthA interactions switch from predominantly subsite-I to subsite-II of the Mtb beta-clamp. Overall, the novel tri-component complex prevents futile ligation activity of MtbLigA on the product of MtbXthA and ensures forward progression of the pathway and productive mycobacterial BER interactions.

Automated summary

A novel tri-component BER complex composed of Mtb beta-clamp, MtbXthA, and MtbLigA has been discovered. In the apo complex, MtbXthA binds simultaneously to subsite-I of Mtb beta-clamp through the 239QLRFPKK245 motif and to MtbLigA through the 104DGQPSWSGKP113 motif. However, in the presence of substrate DNA, the complex adopts a less-extended conformation, and MtbXthA interactions switch from predominantly subsite-I to subsite-II of Mtb beta-clamp. This novel tri-component complex prevents futile ligation activity of MtbLigA on the product of MtbXthA and ensures productive mycobacterial BER interactions.