Mechanism of specific LexA cleavage: Autodigestion and the role of RecA coprotease

Little, John W.

doi:10.1016/0300-9084(91)90108-d

Cited by 328 publications

(258 citation statements)

References 38 publications

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“…The genes induced during the SOS response are repressed in the absence of chromosomal damage by the LexA repressor bound to their promoters (27). LexA autocleavage in the presence of RecA, activated by chromosomal lesions, leads to the derepression of the SOS regulon (28). A reporter gene under the control of a LexA-regulatable promoter identifies mutants elevated for SOS expression, presumably because they experience higher levels of chromosomal lesions.…”

Section: Resultsmentioning

confidence: 99%

RecA-dependent mutants in Escherichia coli reveal strategies to avoid chromosomal fragmentation

Kouzminova

Rotman

Macomber

et al. 2004

Proc. Natl. Acad. Sci. U.S.A.

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RecA-and RecBC-catalyzed repair in eubacteria assembles chromosomes fragmented by double-strand breaks. We propose that recA mutants, being unable to repair fragmented chromosomes, depend on various strategies designed to avoid chromosomal fragmentation. To identify chromosomal fragmentation-avoidance strategies, we screened for Escherichia coli mutants synthetically inhibited in combination with recA inactivation by identifying clones unable to lose a plasmid carrying the recA ؉ gene. Using this screen, we have isolated several RecA-dependent mutants and assigned them to three distinct areas of metabolism. The tdk and rdgB mutants affect synthesis of DNA precursors. The fur, ubiE, and ubiH mutants are likely to have increased levels of reactive oxygen species. The seqA, topA mutants and an insertion in smtA perturbing the downstream mukFEB genes affect nucleoid administration. All isolated mutants show varying degree of SOS induction, indicating elevated levels of chromosomal lesions. As predicted, mutants in rdgB, seqA, smtA, topA, and fur show increased levels of chromosomal fragmentation in recBC mutant conditions. Future characterization of these RecA-dependent mutants will define mechanisms of chromosomal fragmentation avoidance. C hromosomal fragmentation due to double-strand DNA breaks and disintegrated replication forks is a major contributor to genome instability in all organisms (1, 2). The two major pathways used by eukaryotic cells to repair fragmented chromosomes are nonhomologous end joining and homologous recombination (3). Nonhomologous end joining is an imprecise repair, frequently involving loss of genetic information, but it is nevertheless an efficient way to repair double-strand breaks in cells of higher eukaryotes (4). However, disintegrated replication forks, having a single double-strand end, cannot be reassembled by nonhomologous end joining and require error-free recombinational repair (5, 6). The importance of recombinational repair in higher eukaryotes is illustrated by the inviability of recombinational repair mutants in mice (7) and by the rapid death of recombinational repair-defective vertebrate cells due to chromosomal fragmentation (8). In the model eubacterium Escherichia coli, recombinational repair of fragmented chromosomes is catalyzed by the RecA and RecBCD enzymes (9). RecBCD is an exonuclease͞helicase that prepares the doublestrand DNA ends of the break for RecA polymerization (10). RecA filament catalyzes homologous strand exchange of the broken DNA duplex with an intact sister duplex, thus creating an opportunity for double-strand break repair (11).In recA or recBC mutants of E. coli, double-strand DNA breaks are not repaired (12) and cause chromosomal loss and cell death (13,14). However, recA mutant E. coli strains are still 50% viable (15, 16), which indicates that the chromosomal fragmentation is not a frequent event in E. coli and suggests the existence of strategies designed to avoid chromosomal fragmentation. Inactivation of one of these hypothetical avoidance a...

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Section: Resultsmentioning

confidence: 99%

RecA-dependent mutants in Escherichia coli reveal strategies to avoid chromosomal fragmentation

Kouzminova

Rotman

Macomber

et al. 2004

Proc. Natl. Acad. Sci. U.S.A.

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“…But the SOS response is also induced, at least partially, whenever active LexA levels fluctuate downward. In vitro the rate of LexA inactivation increases when the pH becomes slightly alkaline [2]. In vivo LexA is inactivated when cells reach saturation in rich medium [3] and in aging colonies [4].…”

Section: The Sos Responsementioning

confidence: 99%

Stress responses and genetic variation in bacteria

Foster

2005

Mutation Research/Fundamental and Molecular Mechanisms of Mutag

178

106

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Under stressful conditions mechanisms that increase genetic variation can bestow a selective advantage. Bacteria have several stress responses that provide ways in which mutation rates can be increased. These include the SOS response, the general stress response, the heat-shock response, and the stringent response, all of which impact the regulation of error-prone polymerases. Adaptive mutation appears to be process by which cells can respond to selective pressure specifically by producing mutations. In Escherichia coli strain FC40 adaptive mutation involves the following inducible components: (i) a recombination pathway that generates mutations; (ii) a DNA polymerase that synthesizes error-containing DNA; and (iii) stress responses that regulate cellular processes. In addition, a subpopulation of cells enters into a state of hypermutation, giving rise to about 10% of the single mutants and virtually all of the mutants with multiple mutations. These bacterial responses have implications for the development of cancer and other genetic disorders in higher organisms.

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“…Under homeostatic conditions, the transcription factor LexA represses the genes of the SOS response regulon, including itself (40). Upon recognition of DNA damage, LexA is cleaved through autocatalysis promoted by an activated form of RecA, a multifunctional regulator (41). LexA3 (Ind−) is a single amino acid mutant of LexA that is resistant to autocatalytic cleavage and therefore is a potent suppressor of the SOS response (42).…”

Section: Resultsmentioning

confidence: 99%

Tracking, tuning, and terminating microbial physiology using synthetic riboregulators

Callura

Dwyer

Isaacs

et al. 2010

Proc. Natl. Acad. Sci. U.S.A.

173

162

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The development of biomolecular devices that interface with biological systems to reveal new insights and produce novel functions is one of the defining goals of synthetic biology. Our lab previously described a synthetic, riboregulator system that affords for modular, tunable, and tight control of gene expression in vivo. Here we highlight several experimental advantages unique to this RNA-based system, including physiologically relevant protein production, component modularity, leakage minimization, rapid response time, tunable gene expression, and independent regulation of multiple genes. We demonstrate this utility in four sets of in vivo experiments with various microbial systems. Specifically, we show that the synthetic riboregulator is well suited for GFP fusion protein tracking in wildtype cells, tight regulation of toxic protein expression, and sensitive perturbation of stress response networks. We also show that the system can be used for logic-based computing of multiple, orthogonal inputs, resulting in the development of a programmable kill switch for bacteria. This work establishes a broad, easy-to-use synthetic biology platform for microbiology experiments and biotechnology applications. S ynthetic biology seeks to reprogram organisms to alter natural biological behavior or perform novel functions, using engineered gene circuits, pathways, and whole genomes (1-8). Engineered gene circuits, in this regard, are typically constructed by assembling well-characterized biological components into unique architectures to achieve these unnatural capabilities, which are commonly used in biotechnology applications. A need also exists for synthetic biomolecular devices that interface with endogenous systems to track, probe, and influence, rather than reprogram, cellular physiology. Synthetic gene expression platforms that could accomplish this task would be well suited for use in a wide variety of phenotypic and genetic analyses aimed at expanding our knowledge of natural biomolecular components and networks, as well as enhancing our understanding of network dynamics under an array of conditions.In this work, we showcase an engineered riboregulator system that controls gene expression posttranscriptionally through highly specific RNA-RNA interactions (9). RNA molecules are useful components in synthetic biomolecular devices (10, 11) due to their large sequence space, the number of unique structures that can be adopted, the predictability of structure stability, and the range of functions enabled by these structures. Together, these features make RNA molecules ideal for interfacing with biological systems given that they can be used to control gene expression, sense biomolecules, and serve as foundational devices that can perform complex cellular behavior. As such, the engineered, RNA-based device space is remarkably diverse. Other successful examples include independent transcription-translation networks based on orthogonal mRNA-ribosome pairs (12), riboswitches in which RNA aptamers directly control translati...

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Mechanism of specific LexA cleavage: Autodigestion and the role of RecA coprotease

Cited by 328 publications

References 38 publications

RecA-dependent mutants in Escherichia coli reveal strategies to avoid chromosomal fragmentation

RecA-dependent mutants in Escherichia coli reveal strategies to avoid chromosomal fragmentation

Stress responses and genetic variation in bacteria

Tracking, tuning, and terminating microbial physiology using synthetic riboregulators

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