Genetic analysis of an archaeal Holliday junction resolvase in Escherichia coli 1 1Edited by J. Karn

Identification of Escherichia coli ygaQ and rpmG as novel mitomycin C resistance factors implicated in DNA repair

“…The genetic assay we used exploited the extreme MMC sensitivity of an E. coli Δ ruvAB strain ( Figure 1 A) 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 (MMC R ) to Δ ruvAB cells [ 17 ] ( Figure 1 B). An ASKA plasmid library [ 25 ] was transformed into E. coli Δ ruvAB, followed by viability tests on MMC agar, resulting in 21 colonies with apparent MMC R compared with surrounding colonies on replica agar plates, summarized in Figure 1( C).…”

“…In total approximately 11000 of the resultant colonies were master gridded on to LB agar containing chloramphenicol (15 μg/ml), and then replica plated on to LB agar containing either chloramphenicol (15 μg/ml), or chloramphenicol plus MMC (0.2 μg/ml) and IPTG (0.5 mM). A positive control plasmid that gives MMC R in Δ ruvAB E. coli by expressing the resolvase Hjc [ 17 ] was included in every stage to compare to ASKA clones. Note that using Δ ruvAB cells for screening MMC R from the ASKA library was appropriate because ruvA and ruvB genes encoding the RuvAB complex (RuvA 4 or 8 -RuvB 12 ), were on separate ASKA 96-well plates, removing the potential for false-positive MMC R that could arise if from ruvA and ruvB were encoded on the same plasmid.…”

“…Similar activities of RuvABC at blocked replication forks can promote repair of blocking lesions and restart of replication [ 7 , 14 – 16 ]. Δ ruvABC cells can be rescued from MMC sensitivity by expression of alternative Holliday junction nucleases, the archaeal resolvase Hjc [ 17 ], or bacteriophage RusA [ 18 ]. Deletion in E. coli of base excision repair (BER) enzyme MutM and nucleotide excision repair (NER) enzymes UvrABC also cause acute sensitivity to MMC [ 5 ], highlighting how multiple DNA repair roles are be needed to overcome genotoxic effects of MMC.…”

Archaeal Hel308 helicase targets replication forks in vivo and in vitro and unwinds lagging strands

“…The current lack of tools for investigating stalled DNA replication in vivo in homologous archaeal systems led us to heterologous genetics. This approach has been useful for understanding DNA replication in eukaryotes (29), for probing eukaryotic DNA repair pathways with bacterial resolvases (30–32) and for unravelling bacterial repair pathways with an archaeal resolvase (33). We analysed phenotypes of putative helicase genes from the moderately thermophilic archaeon Mth in an E.coli strain carrying a point mutation ( dnaE486 ) in DNA polymerase III.…”

Homologous recombination in the archaea: the means justify the ends

“…Hjc was shown to have properties analogous to the E. coli resolving enzyme RuvC, although the two proteins did not share any sequence similarity. Genetic studies confirmed that expression of an archaeal Hjc gene in E. coli could rescue a ruvC mutant [35]. Subsequently, it was demonstrated that the crenarchaeon S. solfataricus had two distinct resolving enzyme activities, one encoded by the hjc gene and the other named Hje (for HJ endonuclease) which is homologous with Hjc [36,37].…”