Structural and biochemical characterization of the mitomycin C repair exonuclease MrfB

Abstract

Mitomycin C (MMC) repair factor A (mrfA) and factor B (mrfB), encode a conserved helicase and exonuclease that repair DNA damage in the soil-dwelling bacterium Bacillus subtilis. Here we have focused on the characterization of MrfB, a DEDDh exonuclease in the DnaQ superfamily. We solved the structure of the exonuclease core of MrfB to a resolution of 2.1 [A], in what appears to be an inactive state. In this conformation, a predicted -helix containing the catalytic DEDDh residue Asp172 adopts a random coil, which moves Asp172 away from the active site and results in the occupancy of only one of the two catalytic Mg2+ ions. We propose that MrfB resides in this inactive state until it interacts with DNA to become activated. By comparing our structure to an AlphaFold prediction as well as other DnaQ-family structures, we located residues hypothesized to be important for exonuclease function. Using exonuclease assays we show that MrfB is a Mg2+-dependent 3-5 DNA exonuclease. We show that Leu113 aids in coordinating the 3 end of the DNA substrate, and that a basic loop is important for substrate binding. This work provides insight into the function of a recently discovered bacterial exonuclease important for the repair of MMC-induced DNA adducts. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=111 SRC="FIGDIR/small/580553v1_ufig1.gif" ALT="Figure 1"> View larger version (27K): org.highwire.dtl.DTLVardef@ac964borg.highwire.dtl.DTLVardef@bf1f81org.highwire.dtl.DTLVardef@1ad4e89org.highwire.dtl.DTLVardef@ff0641_HPS_FORMAT_FIGEXP M_FIG C_FIG