Genetic Requirements for High Constitutive SOS Expression in<i>recA730</i>Mutants of Escherichia coli

Vlašić, Ignacija; Šimatović, Ana; Brčić-Kostić, Krunoslav

doi:10.1128/jb.00368-11

Cited by 9 publications

(8 citation statements)

References 46 publications

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“…In the present study we found that, contrary to RecQ deletion, the recB1080 mutation drastically reduces the survival of E . coli cells upon UV radiation ( Fig 3 ) in line with previous results [ 5 , 26 ]. Importantly, in the recB1080 background, the lack of RecQ DNA unwinding activity ( ΔrecQ , recQ-dWH ) caused further reduction of UV survival, whereas this effect was not seen in strains expressing unwinding-competent RecQ constructs ( recQ* , recQ-dH ) ( Fig 3 ).…”

Section: Resultssupporting

confidence: 92%

“…Previously we and others found that mutations in the recQ gene do not affect the sensitivity of E . coli cells to UV irradiation [ 5 , 15 , 25 , 26 ]. In the present study we found that, contrary to RecQ deletion, the recB1080 mutation drastically reduces the survival of E .…”

Section: Resultsmentioning

confidence: 99%

“…Recent biochemical/biophysical investigations of the DNA-restructuring activities of RecBCD and RecQ enzymes yielded important insights into their mechanisms of action, and precisely defined the mechanistic perturbations characteristic of their engineered variants [ 5 , 15 , 25 , 26 , 30 ]. Specifically, the enhanced helicase but deficient nuclease and RecA-loading activities of the RecB 1080 CD complex lead to the formation of abnormally long, partially RecA-filled 5’ and 3’ DNA overhangs [ 6 , 31 ] (upper row in Fig 6 ), while the enhanced (highly processive) DNA unwinding but compromised DNA invasion disruption and/or shuttling activities of the RecQ* and RecQ-dH variants lead to impaired suppression of IR events [ 15 ].…”

Section: Discussionmentioning

confidence: 99%

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Functional fine-tuning between bacterial DNA recombination initiation and quality control systems

et al. 2018

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Homologous recombination (HR) is crucial for the error-free repair of DNA double-strand breaks (DSBs) and the restart of stalled replication. However, imprecise HR can lead to genome instability, highlighting the importance of HR quality control. After DSB formation, HR proceeds via DNA end resection and recombinase loading, whereas helicase-catalyzed disruption of a subset of subsequently formed DNA invasions is thought to be essential for maintaining HR accuracy via inhibiting illegitimate (non-allelic) recombination. Here we show that in vitro characterized mechanistic aberrations of E. coli RecBCD (resection and recombinase loading) RecQ (multifunctional DNA-restructuring helicase) mutant enzyme variants, on one hand, cumulatively deteriorate cell survival under certain conditions of genomic stress. On the other hand, we find that RecBCD and RecQ defects functionally compensate each other in terms of HR accuracy. The abnormally long resection and unproductive recombinase loading activities of a mutant RecBCD complex (harboring the D1080A substitution in RecB) cause enhanced illegitimate recombination. However, this compromised HR-accuracy phenotype is suppressed in double mutant strains harboring mutant RecQ variants with abnormally enhanced helicase and inefficient invasion disruptase activities. These results frame an in vivo context for the interplay of biochemical activities leading to illegitimate recombination, and underscore its long-range genome instability effects manifest in higher eukaryotes.

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Section: Resultssupporting

confidence: 92%

Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

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Functional fine-tuning between bacterial DNA recombination initiation and quality control systems

et al. 2018

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“…Under these conditions, the SOS regulon is partially derepressed and much of the temporal regulation normally imposed on pol V is circumvented: pol V is constitutively formed through UmuD cleavage and activated to pol V Mut. However, the ability of RecA(E38K) to spontaneously form nucleoprotein filaments depends upon the cellular concentration of the RecA mutant [ 15 ] and RecA(E38K) can be further activated under various conditions [ 18 ]. Indeed, UV treatment results in the apparent induction of UmuC in the recA (E38K) lexA + background, where all LexA-regulated proteins are expected to already be expressed in the absence of DNA damage.…”

Section: Resultsmentioning

confidence: 99%

Regulation of Mutagenic DNA Polymerase V Activation in Space and Time

et al. 2015

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Spatial regulation is often encountered as a component of multi-tiered regulatory systems in eukaryotes, where processes are readily segregated by organelle boundaries. Well-characterized examples of spatial regulation are less common in bacteria. Low-fidelity DNA polymerase V (UmuD′2C) is produced in Escherichia coli as part of the bacterial SOS response to DNA damage. Due to the mutagenic potential of this enzyme, pol V activity is controlled by means of an elaborate regulatory system at transcriptional and posttranslational levels. Using single-molecule fluorescence microscopy to visualize UmuC inside living cells in space and time, we now show that pol V is also subject to a novel form of spatial regulation. After an initial delay (~ 45 min) post UV irradiation, UmuC is synthesized, but is not immediately activated. Instead, it is sequestered at the inner cell membrane. The release of UmuC into the cytosol requires the RecA* nucleoprotein filament-mediated cleavage of UmuD→UmuD′. Classic SOS damage response mutants either block [umuD(K97A)] or constitutively stimulate [recA(E38K)] UmuC release from the membrane. Foci of mutagenically active pol V Mut (UmuD′2C-RecA-ATP) formed in the cytosol after UV irradiation do not co-localize with pol III replisomes, suggesting a capacity to promote translesion DNA synthesis at lesions skipped over by DNA polymerase III. In effect, at least three molecular mechanisms limit the amount of time that pol V has to access DNA: (1) transcriptional and posttranslational regulation that initially keep the intracellular levels of pol V to a minimum; (2) spatial regulation via transient sequestration of UmuC at the membrane, which further delays pol V activation; and (3) the hydrolytic activity of a recently discovered pol V Mut ATPase function that limits active polymerase time on the chromosomal template.

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“…The reason for such analysis lies in the fact that the level of mutagenesis promoted by pol V differs considerably in the various strain backgrounds and this variability is likely caused by the extent of endogenous single-stranded DNA in the cell, which is an important factor in hyper-activating RecA730 and promoting pol V-induced mutagenesis in vivo [36, 38]. Since the only known biochemical difference between UmuC_Y11A and wild-type pol V is its ability to incorporate riboncleotides [2], by expressing umuC_ Y11A-dependent mutagenesis as a percent of the mutagenesis induced by wild-type pol V, we are able to focus only on those mutagenic events that are specifically related to rNMP incorporation and repair.…”

Section: Resultsmentioning

confidence: 99%

Investigating the mechanisms of ribonucleotide excision repair in Escherichia coli

Vaisman

McDonald

Noll

et al. 2014

Mutation Research/Fundamental and Molecular Mechanisms of Mutag

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Low fidelity Escherichia coli DNA polymerase V (pol V/UmuD′2C) is best characterized for its ability to perform translesion synthesis (TLS). However, in recA730 lexA(Def) strains, the enzyme is expressed under optimal conditions allowing it to compete with the cell’s replicase for access to undamaged chromosomal DNA and leads to a substantial increase in spontaneous mutagenesis. We have recently shown that a Y11A substitution in the “steric gate” residue of UmuC reduces both base and sugar selectivity of pol V, but instead of generating an increased number of spontaneous mutations, strains expressing umuC_Y11A are poorly mutable in vivo. This phenotype is attributed to efficient RNase HII-initiated repair of the misincorporated ribonucleotides that concomitantly removes adjacent misincorporated deoxyribonucleotides. We have utilized the ability of the pol V steric gate mutant to promote incorporation of large numbers of errant ribonucleotides into the E. coli genome to investigate the fundamental mechanisms underlying ribonucleotide excision repair (RER). Here, we demonstrate that RER is normally facilitated by DNA polymerase I (pol I) via classical “nick translation”. In vitro, pol I displaces 1–3 nucleotides of the RNA/DNA hybrid and through its 5′→3′ (exo/endo) nuclease activity releases ribo- and deoxyribonucleotides from DNA. In vivo, umuC_Y11A-dependent mutagenesis changes significantly in polymerase-deficient, or proofreading-deficient polA strains, indicating a pivotal role for pol I in ribonucleotide excision repair (RER). However, there is also considerable redundancy in the RER pathway in E. coli. Pol I’s strand displacement and FLAP- exo/endonuclease activities can be facilitated by alternate enzymes, while the DNA polymerization step can be assumed by high-fidelity pol III. We conclude that RNase HII and pol I normally act to minimize the genomic instability that is generated through errant ribonucleotide incorporation, but that the “nick-translation” activities encoded by the single pol I polypeptide can be undertaken by a variety of back-up enzymes.

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Genetic Requirements for High Constitutive SOS Expression inrecA730Mutants of Escherichia coli

Cited by 9 publications

References 46 publications

Functional fine-tuning between bacterial DNA recombination initiation and quality control systems

Functional fine-tuning between bacterial DNA recombination initiation and quality control systems

Regulation of Mutagenic DNA Polymerase V Activation in Space and Time

Investigating the mechanisms of ribonucleotide excision repair in Escherichia coli

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