Characterization of DnaB-DnaG Interaction in M. tuberculosis Using Small-Angle X-ray Scattering-Based Dissociation...

Area of Science:

• Molecular Biology
• Biochemistry
• Structural Biology

Background:

• DNA replication is essential for bacterial survival and proliferation.
• The replisome, comprising proteins like helicase and primase, orchestrates DNA replication.
• Mycobacterium tuberculosis (Mtb) possesses unique replication machinery, making it a target for novel therapeutics.

Purpose of the Study:

• To investigate the intricate interactions between helicase and primase in M. tuberculosis.
• To characterize the binding dynamics and structural interplay of these key replication proteins.
• To identify potential therapeutic targets for inhibiting DNA replication in M. tuberculosis.

Main Methods:

• Purification of complementary protein domains of helicase and primase.
• Surface Plasmon Resonance (SPR) analysis to quantify binding kinetics.
• Cross-linking assays to detect protein complex formation.
• Small-angle X-ray scattering (SAXS) to study solution dynamics.

Main Results:

• SPR analysis yielded a binding dissociation constant (Kd) of 0.21 ± 0.08 µM, indicating significant interaction.
• Cross-linking assays suggested the formation of a helicase-primase heterodimer.
• SAXS dissociation assays provided insights into the dynamic interactions in solution.

Conclusions:

• Helicase and primase form a stable complex crucial for M. tuberculosis DNA replication.
• The characterized structural interplay reveals potential vulnerabilities in the Mtb replisome.
• These findings pave the way for developing targeted inhibitors of DNA replication in M. tuberculosis.