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

Abstract

The mycobacterial mutasome which is minimally comprised of ImuA′, ImuB, and DnaE2 proteins — has been implicated in DNA damage–induced mutagenesis in <em>Mycobacterium tuberculosis</em>. ImuB, predicted to enable mutasome function via its interaction with the β 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 <em>imuB</em> 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.

A nucleoid-associated protein is involved in the emergence of antibiotic resistance by promoting the frequent exchange of the replicative DNA polymerase in Mycobacterium smegmatis

“…ImuB recruits ImuA’ and DnaE2 into an active complex by binding to the β-clamp subunit of the replisome. ImuA’ and DnaE2 bind ImuB but not the β-clamp and therefore require ImuB to associate with the replisome and catalyze mutations ( 8 , 11 , 28 , 29 ). Despite being co-transcribed, ImuA’-mScarlet and mScarlet-ImuB had dramatically different fluorescence intensities: ImuA’-mScarlet was barely detectable above background fluorescence levels, while mScarlet-ImuB was brightly fluorescent (Fig.…”

A nucleoid-associated protein is involved in the emergence of antibiotic resistance by promoting the frequent exchange of the replicative DNA polymerase inM. smegmatis

“…ImuB recruits ImuA’ and DnaE2 into an active complex by binding to the β-clamp subunit of the replisome. ImuA’ and DnaE2 bind ImuB but not the β-clamp, and therefore require ImuB to associate with the replisome and catalyze mutations (8, 11, 27, 28). Despite being co-transcribed, ImuA’-mScarlet and mScarlet-ImuB had dramatically different fluorescence intensities: ImuA’-mScarlet was barely detectable above background fluorescence levels, while mScarlet-ImuB was brightly fluorescent ( Fig.…”