The RecA-NT homology motif in ImuB mediates the interaction with ImuA' which is essential for DNA damage-induced mutagenesis

• Published in: The Journal of biological chemistry 2024-12, p.108108
• Main Authors: Santos, Joana A, Timinskas, Kęstutis, Ramudzuli, Atondaho A, Lamers, Meindert H, Venclovas, Česlovas, Warner, Digby F, Gessner, Sophia J
• Format: Article
• Language: English
• ISSN: 1083-351X; 1083-351X
• DOI: 10.1016/j.jbc.2024.108108

The mycobacterial mutasome - comprising ImuA', ImuB, and DnaE2 - has been implicated in DNA damage-induced mutagenesis in Mycobacterium tuberculosis. ImuB, which is predicted to enable mutasome function via its interaction with the β clamp, is a catalytically inactive Y-family DNA polymerase. Like some other members of the Y-family, ImuB features a recently identified amino acid motif with homology to the RecA N-terminus (RecA-NT). Given the role of RecA-NT in RecA oligomerization, we hypothesized that ImuB RecA-NT mediates the interaction with ImuA', a RecA homolog of unknown function. Here, we constructed a panel of imuB alleles in which the RecA-NT was removed, or mutated. Our results indicate that RecA-NT is critical for the interaction of ImuB with ImuA'. A region downstream of RecA-NT, ImuB-C, appears to stabilize the ImuB-ImuA' interaction, but its removal does not prevent complex formation. In contrast, replacing two hydrophobic residues of RecA-NT, L378 and V383, disrupts the ImuA'-ImuB interaction. To our knowledge, this is the first experimental evidence suggesting a role for RecA-NT in mediating the interaction between a Y-family member and a RecA homolog.The mycobacterial mutasome - comprising ImuA', ImuB, and DnaE2 - has been implicated in DNA damage-induced mutagenesis in Mycobacterium tuberculosis. ImuB, which is predicted to enable mutasome function via its interaction with the β clamp, is a catalytically inactive Y-family DNA polymerase. Like some other members of the Y-family, ImuB features a recently identified amino acid motif with homology to the RecA N-terminus (RecA-NT). Given the role of RecA-NT in RecA oligomerization, we hypothesized that ImuB RecA-NT mediates the interaction with ImuA', a RecA homolog of unknown function. Here, we constructed a panel of imuB alleles in which the RecA-NT was removed, or mutated. Our results indicate that RecA-NT is critical for the interaction of ImuB with ImuA'. A region downstream of RecA-NT, ImuB-C, appears to stabilize the ImuB-ImuA' interaction, but its removal does not prevent complex formation. In contrast, replacing two hydrophobic residues of RecA-NT, L378 and V383, disrupts the ImuA'-ImuB interaction. To our knowledge, this is the first experimental evidence suggesting a role for RecA-NT in mediating the interaction between a Y-family member and a RecA homolog.