Abstract
Using the ASKA (A Complete Set of Escherichia coli K-12 ORF Archive) library for genome-wide screening of E. coli proteins we identified that expression of ygaQ and rpmG promotes mitomycin C resistance (MMC(R)). YgaQ mediated MMC(R) was independent of homologous recombination involving RecA or RuvABC, but required UvrD. YgaQ is an uncharacterized protein homologous with α-amylases that we identified to have nuclease activity directed to ssDNA of 5' flaps. Nuclease activity was inactivated by mutation of two amino acid motifs, which also abolished MMC(R). RpmG is frequently annotated as a bacterial ribosomal protein, although forms an operon with MutM glycosylase and a putative deubiquitinating (DUB) enzyme, YicR. RpmG associated MMC(R) was dependent on MutM. MMC(R) from RpmG resembles DNA repair phenotypes reported for 'idiosyncratic ribosomal proteins' in eukaryotes.
Highlights
Chemicals causing covalent modifications to DNA are cytotoxic when their products interfere with biological processes including DNA replication and gene transcription
Identification of ygaQ and rpmG as mitomycin C resistance factors in E. coli We searched for uncharacterized genes in E. coli whose expression overcame growth inviability associated with Mitomycin C (MMC) induced DNA damage
The genetic assay we used exploited the extreme MMC sensitivity of an E. coli ruvAB strain (Figure 1A) resulting from it lacking the RuvABC DNA repair complex. This followed a rationale from previous work identifying that the archaeal Holliday junction resolvase Hjc can restore mitomycin C resistance (MMCR) to ruvAB cells [17] (Figure 1B)
Summary
Chemicals causing covalent modifications to DNA are cytotoxic when their products interfere with biological processes including DNA replication and gene transcription. Mitomycin C (MMC) provokes interstrand DNA cross-links at 5 -GNC-3 or 5 -CG-3 sequences, and mono-adducts at guanine bases [1,2,3]. It is a natural antimicrobial synthesized by Streptomyces caespitosis that is effective as a treatment for human cancers, and there is continuing interest in mechanisms cells use to overcome genotoxic damage associated with MMC and other cross-linkers [4]. Removal and repair of MMC induced DNA damage, involves interplay between nucleotide excision repair, homologous recombination and repair polymerases. The exact events post-excision of the lesion probably depend on the context of repair and the type of lesion being removed
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