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bioRxiv · 10.1101/2023.03.28.534377

RecA-NT homology motif in ImuB is essential for mycobacterial ImuA'-ImuB protein interaction and mutasome function

Santos, J. A.·Timinskas, K.·Lamers, M. H.·Venclovas, C.·Warner, D. F.·Gessner, S. J.
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Abstract

The mycobacterial mutasome - minimally comprising ImuA, ImuB, and DnaE2 proteins - has been implicated in DNA damage-induced mutagenesis in Mycobacterium tuberculosis. ImuB, predicted to enable mutasome function via its interaction with the {beta} clamp, is a catalytically inactive member of the Y-family of DNA polymerases. Like other members of the Y family, ImuB features a recently identified amino acid motif with homology to the RecA-N-terminus (RecA-NT). In RecA, the motif mediates oligomerization of RecA monomers into RecA filaments. Given the role of ImuB in the mycobacterial mutasome, we hypothesized that the ImuB RecA-NT motif might mediate its interaction with ImuA, a RecA homolog of unknown function. To investigate this possibility, we constructed a panel of imuB alleles in which RecA-NT was removed, or mutated. Results from microbiological and biochemical assays indicate that RecA-NT is critical for the interaction of ImuB with ImuA. A region downstream of RecA-NT (ImuB-C) also appears to stabilize the ImuB-ImuA interaction, but its removal does not prevent complex formation. In contrast, replacing two key hydrophobic residues of RecA-NT, L378 and V383, is sufficient to disrupt ImuA-ImuB interaction. To our knowledge, this constitutes the first experimental evidence showing the role of the RecA-NT motif in mediating the interaction between a Y-family member and a RecA homolog.

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Santos, J. A., Timinskas, K., Lamers, M. H., Venclovas, C., Warner, D. F., Gessner, S. J.. 2023-03-28. RecA-NT homology motif in ImuB is essential for mycobacterial ImuA'-ImuB protein interaction and mutasome function. https://doi.org/10.1101/2023.03.28.534377

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