Structural and biochemical characterization of the mitomycin C repair exonuclease MrfB.
Nucleic Acids Res
; 52(11): 6347-6359, 2024 Jun 24.
Article
em En
| MEDLINE
| ID: mdl-38661211
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 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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Base de dados:
MEDLINE
Assunto principal:
Bacillus subtilis
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Proteínas de Bactérias
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Mitomicina
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Magnésio
Idioma:
En
Ano de publicação:
2024
Tipo de documento:
Article