RecJ nuclease is required for SOS induction after introduction of a double-strand break in a RecA loading deficient recB mutant of Escherichia coli

Vlašić, Ignacija; Ivančić-Baće, Ivana; Imešek, Mirna; Mihaljevic, Boris; Brčić-Kostić, Krunoslav

doi:10.1016/j.biochi.2008.04.002

Cited by 13 publications

(12 citation statements)

References 30 publications

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“…The group of proteins that was exclusively up‐regulated after 6, 12 and 24 h ciprofloxacin treatment (see also Figure 3 cluster ‘b’), contains mainly proteins involved in transcriptional and translational processes, like DNA mismatch, RNA polymerization, aminoacyl‐tRNA synthesis, purine and pyrimidine metabolism, as well as the secretion system and the SOS response (Fonville et al , 2010), like recombinase A and J. These data indicate that the cells are trying to compensate the DNA‐topo‐isomeric stress induced by the ciprofloxacin treatment (Michel, 2005; Cirz et al , 2007; López et al , 2007; Vlasić et al , 2008). Intriguingly, we also found the protein TetR in this cluster, which was recently found to be specifically mutated in ciprofloxacin‐resistant strains of Bacillus anthracis (Serizawa et al , 2010), underlining the relevance of the specific protein changes detected.…”

Section: Resultsmentioning

confidence: 97%

Absolute quantification of microbial proteomes at different states by directed mass spectrometry

Schmidt

Beck

Malmström

et al. 2011

Molecular Systems Biology

112

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The developed, directed mass spectrometry workflow allows to generate consistent and system-wide quantitative maps of microbial proteomes in a single analysis. Application to the human pathogen L. interrogans revealed mechanistic proteome changes over time involved in pathogenic progression and antibiotic defense, and new insights about the regulation of absolute protein abundances within operons.

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Section: Resultsmentioning

confidence: 97%

Absolute quantification of microbial proteomes at different states by directed mass spectrometry

Schmidt

Beck

Malmström

et al. 2011

Molecular Systems Biology

112

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“…In the absence of RecB, RecJ single-strand-dependent exonuclease appears to be able to prepare DSEs for RecA filament formation and, in cooperation with a DNA helicase, allows induction of an SOS response (Vlasic et al 2008) and some recombination (Ivancic-Bace et al 2005). To test whether activation of SOS causes most of the sensitivity of Δ recB cells to TLD, we asked whether Δ recB hyper-TLD is RecA dependent.…”

Section: Resultsmentioning

confidence: 99%

Pathways of Resistance to Thymineless Death in Escherichia coli and the Function of UvrD

Fonville

Vaksman²,

DeNapoli

et al. 2011

Genetics

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Thymineless death (TLD) is the rapid loss of viability in bacterial, yeast, and human cells starved of thymine. TLD is the mode of action of common anticancer drugs and some antibiotics. TLD in Escherichia coli is accompanied by blocked replication and chromosomal DNA loss and recent work identified activities of recombination protein RecA and the SOS DNA-damage response as causes of TLD. Here, we examine the basis of hypersensitivity to thymine deprivation (hyper-TLD) in mutants that lack the UvrD helicase, which opposes RecA action and participates in some DNA repair mechanisms, RecBCD exonuclease, which degrades double-stranded linear DNA and works with RecA in double-strand-break repair and SOS induction, and RuvABC Holliday-junction resolvase. We report that hyper-TLD in ∆uvrD cells is partly RecA dependent and cannot be attributed to accumulation of intermediates in mismatch repair or nucleotide-excision repair. These data imply that both its known role in opposing RecA and an additional as-yet-unknown function of UvrD promote TLD resistance. The hyper-TLD of ∆ruvABC cells requires RecA but not RecQ or RecJ. The hyper-TLD of recB cells requires neither RecA nor RecQ, implying that neither recombination nor SOS induction causes hyper-TLD in recB cells, and RecQ is not the sole source of double-strand ends (DSEs) during TLD, as previously proposed; models are suggested. These results define pathways by which cells resist TLD and suggest strategies for combating TLD resistance during chemotherapies.

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“…In order to determine whether 5Ј-3Ј exonuclease activity is required for cSOS expression in the recA730 mutants, we studied cSOS expression in wt, recB1080, and recB21 backgrounds. It was shown that RecJ nuclease is crucial for recombination (13), SOS induction after introduction of DSBs (38), and cSOS expression in a recB1080 mutant (14). Figure 3A shows that the recA730 recJ double mutant had high cSOS expression, even higher than that of the recA730 single mutant.…”

Section: It Was Recently Shown That Csos Expression In a Reca4142mentioning

confidence: 97%

Genetic Requirements for High Constitutive SOS Expression inrecA730Mutants of Escherichia coli

2011

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The RecA protein in its functional state is in complex with single-stranded DNA, i.e., in the form of a RecA filament. In SOS induction, the RecA filament functions as a coprotease, enabling the autodigestion of the LexA repressor. The RecA filament can be formed by different mechanisms, but all of them require three enzymatic activities essential for the processing of DNA double-stranded ends. These are helicase, 5-3 exonuclease, and RecA loading onto single-stranded DNA (ssDNA). In some mutants, the SOS response can be expressed constitutively during the process of normal DNA metabolism. The RecA730 mutant protein is able to form the RecA filament without the help of RecBCD and RecFOR mediators since it better competes with the single-strand binding (SSB) protein for ssDNA. As a consequence, the recA730 mutants show high constitutive SOS expression. In the study described in this paper, we studied the genetic requirements for constitutive SOS expression in recA730 mutants. Using a ␤-galactosidase assay, we showed that the constitutive SOS response in recA730 mutants exhibits different requirements in different backgrounds. In a wild-type background, the constitutive SOS response is partially dependent on RecBCD function. In a recB1080 background (the recB1080 mutation retains only helicase), constitutive SOS expression is partially dependent on RecBCD helicase function and is strongly dependent on RecJ nuclease. Finally, in a recB-null background, the constitutive SOS expression of the recA730 mutant is dependent on the RecJ nuclease. Our results emphasize the importance of the 5-3 exonuclease for high constitutive SOS expression in recA730 mutants and show that RecBCD function can further enhance the excellent intrinsic abilities of the RecA730 protein in vivo.The RecA protein is a central component of the recombination machinery which is required for double-strand break (DSB) repair and for producing genetic variation during conjugation in bacteria and meiosis in eukaryotes. An additional role of the RecA protein is in the induction of an SOS response. In order to exhibit its biological functions, the RecA protein must be in the form of a RecA-single-stranded DNA (ssDNA) filament (RecA filament). The RecA filament is formed after processing of DNA damage, i.e., DSBs and single-stranded gaps (SSGs). In wild-type (wt) Escherichia coli strains, DSBs are processed into RecA filaments by the RecBCD pathway of recombination, whereas SSGs utilize the RecF recombination pathway (16,17). DSBs can also be processed into RecA filaments by the RecF recombination pathway when the RecBCD enzyme is missing or is inactive, as is the case in a mutant recBC sbcBC(D) strain with multiple mutations (17). Three essential enzymatic activities are required for the processing of DSBs and subsequent RecA filament formation: helicase, 5Ј-3Ј exonuclease, and RecA loading onto ssDNA. After interaction with a Chi site (GCTGGT GG), the RecBCD enzyme exhibits all of these activities, whereas within the RecF recombination machinery, the pa...

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RecJ nuclease is required for SOS induction after introduction of a double-strand break in a RecA loading deficient recB mutant of Escherichia coli

Cited by 13 publications

References 30 publications

Absolute quantification of microbial proteomes at different states by directed mass spectrometry

Absolute quantification of microbial proteomes at different states by directed mass spectrometry

Pathways of Resistance to Thymineless Death in Escherichia coli and the Function of UvrD

Genetic Requirements for High Constitutive SOS Expression inrecA730Mutants of Escherichia coli

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