Structural and biochemical characterization of the mitomycin C repair exonuclease MrfB

Manthei, Kelly A.; Munson, Lia M.; Nandakumar, Jayakrishnan; Simmons, Lyle A.

doi:10.1093/nar/gkae308

Title: Structural and biochemical characterization of the mitomycin C repair exonuclease MrfB

Journal Article · 25 April 2024 · Nucleic Acids Research

DOI: https://doi.org/10.1093/nar/gkae308 · OSTI ID:2470249

Manthei, Kelly A. ^[1]; Munson, Lia M. ^[1]; Nandakumar, Jayakrishnan ^[1];

^[1]

Univ. of Michigan, Ann Arbor, MI (United States)

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 Å, in what appears to be an inactive state. In this conformation, a predicted α-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.

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Research Organization:: Argonne National Laboratory (ANL), Argonne, IL (United States). Advanced Photon Source (APS)

Sponsoring Organization:: Michigan Economic Development Corporation; Michigan Technology Tri-Corridor; National Institutes of Health (NIH); USDOE Office of Science (SC), Basic Energy Sciences (BES). Scientific User Facilities (SUF)

Grant/Contract Number:: AC02-06CH11357

OSTI ID:: 2470249

Journal Information:: Nucleic Acids Research, Journal Name: Nucleic Acids Research Journal Issue: 11 Vol. 52; ISSN 0305-1048

Publisher:: Oxford University PressCopyright Statement

Country of Publication:: United States

Language:: English

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