Chromosomal Fragmentation Is the Major Consequence of the <i>rdgB</i> Defect in <i>Escherichia coli</i>

Lukas, Lisa; Kuzminov, Andrei

doi:10.1534/genetics.105.051144

Cited by 22 publications

(30 citation statements)

References 16 publications

(18 reference statements)

Supporting

Mentioning

Contrasting

Order By: Relevance

“…This also lends strong genetic support to the results of physical studies indicating that uracil incorporation and excision cause double-strand breaks in the E. coli chromosome (34,35,37). The number of isolated inserts, together with a similar number from the earlier search for RdgB-dependent mutants (45), also argues against the existence of other, nonessential genes involved in double-strand break repair in E. coli besides recA, recBC, and ruvABC. In fact, the combined density of inserts in these six genes from the two screens is 44 inserts/10,119 bp, or 1 insert per 230 bp, making failure of detection of any but a very small gene unlikely.…”

Section: Vol 190 2008 Dut-dependent Mutants In E Coli 5843supporting

confidence: 64%

“…Using pRL27 effectively limits the transposon to a single hop, because the transposase gene is outside of the insertional cassette. Also, our significant collection of the pRL27-induced mutations from this and other projects (10,34,37,45,61) demonstrates that the mutant phenotypes are not limited to complete gene inactivation, but also include polar effects on the downstream genes and antisense effects on the upstream genes. Infrequently, we even encounter inactivation of the regulatory C-terminal domains of proteins, as well as gene overexpression (a particular orientation of the insert in the promoter region).…”

Section: Methodsmentioning

confidence: 82%

“…The strain L-44 pEAK12-4 (later on with pHT2, as well) was mutagenized, plated on MacConkey-lactose plates supplemented with 10 g/ml kanamycin to obtain about 200 to 300 transformants per plate, and incubated at 35.5°C for 36 to 48 h (37,45). MacConkey medium allows Lac ϩ cells to form purple colonies and Lac Ϫ cells to form pale colonies.…”

Section: Methodsmentioning

confidence: 99%

“…At 35.5°C, pEAK12-4 replicates less efficiently and is lost from cells at a rate of ϳ5% per generation. Since it takes ϳ25 generations to form a regular-size (3-to 4-mm) colony, this apparently very low rate of plasmid loss translates into a colony in which only ϳ30% of the cells still keep the plasmid (0.95 25 ϳ 0.3), which results in the sectoring phenotype of the colonies (a purple "star" in the center surrounded by pale borders) (37,45). At 42°C, L-44 loses pEAK12-4 completely and forms pale colonies on MacConkey agar.…”

Section: Methodsmentioning

confidence: 99%

See 3 more Smart Citations

Synthetic Lethality with the dut Defect in Escherichia coli Reveals Layers of DNA Damage of Increasing Complexity Due to Uracil Incorporation

2008

Self Cite

View full text Add to dashboard Cite

Synthetic lethality is inviability of a double-mutant combination of two fully viable single mutants, commonly interpreted as redundancy at an essential metabolic step. The dut-1 defect in Escherichia coli inactivates dUTPase, causing increased uracil incorporation in DNA and known synthetic lethalities [SL(dut) mutations]. According to the redundancy logic, most of these SL(dut) mutations should affect nucleotide metabolism. After a systematic search for SL(dut) mutants, we did identify a single defect in the DNA precursor metabolism, inactivating thymidine kinase (tdk), that confirmed the redundancy explanation of synthetic lethality. However, we found that the bulk of mutations interacting genetically with dut are in DNA repair, revealing layers of damage of increasing complexity that uracil-DNA incorporation sends through the chromosomal metabolism. Thus, we isolated mutants in functions involved in (i) uracil-DNA excision (ung, polA, and xthA); (ii) doublestrand DNA break repair (recA, recBC, and ruvABC); and (iii) chromosomal-dimer resolution (xerC, xerD, and ftsK). These mutants in various DNA repair transactions cannot be redundant with dUTPase and instead reveal "defect-damage-repair" cycles linking unrelated metabolic pathways. In addition, two SL(dut) inserts (phoU and degP) identify functions that could act to support the weakened activity of the Dut-1 mutant enzyme, suggesting the "compensation" explanation for this synthetic lethality. We conclude that genetic interactions with dut can be explained by redundancy, by defect-damage-repair cycles, or as compensation.

show abstract

Section: Vol 190 2008 Dut-dependent Mutants In E Coli 5843supporting

confidence: 64%

Section: Methodsmentioning

confidence: 82%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

See 2 more Smart Citations

Synthetic Lethality with the dut Defect in Escherichia coli Reveals Layers of DNA Damage of Increasing Complexity Due to Uracil Incorporation

2008

Self Cite

View full text Add to dashboard Cite

show abstract

“…Rather, the main function of RdgB seems to be to prevent Endo V-triggered chromosomal fragmentation (Fig. 8), as suggested by (i) the unviability of rdgB recA, rdgB recBC, and rdgB ruv double mutants (5,9,33); (ii) the significant chromosomal fragmentation in rdgB recBC double mutants (5,25); and (iii) the suppression of both unviability and chromosomal fragmentation by inactivation of Endo V (5; Budke and Kuzminov, unpublished data). It remains to be tested whether the rdgB defect actually increases the amount of dITP and dXTP in the DNA precursor pools and whether the Endo V-recognized modifications in the DNA of rdgB mutants are indeed hypoxanthines and xanthines.…”

mentioning

confidence: 99%

Hypoxanthine Incorporation Is Nonmutagenic in Escherichia coli

Budke

Kuzminov

2006

J Bacteriol

Self Cite

View full text Add to dashboard Cite

Endonuclease V, encoded by the nfi gene, initiates removal of the base analogs hypoxanthine and xanthine from DNA, acting to prevent mutagenesis from purine base deamination within the DNA. On the other hand, the RdgB nucleotide hydrolase in Escherichia coli is proposed to prevent hypoxanthine and xanthine incorporation into DNA by intercepting the noncanonical DNA precursors dITP and dXTP. Because many base analogs are mutagenic when incorporated into DNA, it is intuitive to think of RdgB as acting to prevent similar mutagenesis from deaminated purines in the DNA precursor pools. To test this idea, we used a set of Claire Cupples' strains to detect changes in spontaneous mutagenesis spectra, as well as in nitrous acid-induced mutagenesis spectra, in wild-type cells and in rdgB single, nfi single, and rdgB nfi double mutants. We found neither a significant increase in spontaneous mutagenesis in rdgB and nfi single mutants or the double mutant nor any changes in nitrous acid-induced mutagenesis for rdgB mutant strains. We conclude that incorporation of deaminated purines into DNA is nonmutagenic.Three of the four DNA bases-cytosine, adenine, and guanine-have amino groups. Deamination of these three bases ( Fig. 1) generates the base analogs uracil, hypoxanthine, and xanthine, which have pairing specificities different from the original bases. Therefore, the cell employs excision repair to prevent mutagenesis due to spontaneous DNA base deamination. For example, in Escherichia coli, uracil-DNA glycosylase encoded by the ung gene initiates excision of uracils from DNA (12, 30). The ung mutants experience a 30-fold increase in GC3AT transitions (13), owing to the deamination of cytosine to uracil in the DNA (31). Deamination of adenine to hypoxanthine in the DNA (24) is also known to cause mutations: hypoxanthine preferentially base pairs with cytosine (3,18,35,39,51), and its formation in DNA, for example, as a result of nitrous acid exposure, leads to AT3GC transitions (45). Hypoxanthine removal from DNA in E. coli is initiated by the product of the nfi gene, endonuclease V (Endo V) (53, 54), and, to a much lesser extent, by the product of the alkA gene, 3-methyladenine-DNA glycosylase (43,44). Although nfi mutants do not show increased spontaneous mutagenesis, they are more mutable by nitrous acid (45), an agent known to promote deamination of DNA bases (55).Bases can deaminate not only in DNA but in nucleotides as well, both chemically (32, 46) and enzymatically (56). Deamination of DNA precursors should lead to accumulation of noncanonical nucleoside triphosphates in the cell's DNA precursor pool. These noncanonical nucleotides, such as dITP, dXTP, and dUTP, can be incorporated into the newly synthesized DNA, albeit less efficiently than regular DNA precursors (3,38,42,51). Noncanonical DNA precursors are actively intercepted and removed from the DNA precursor pool. For example, dUTP is hydrolyzed to dUMP by deoxyuridine triphosphatase (2, 16), encoded by the dut gene (19). Indirect evidence (5; B. Budke and A....

show abstract

Modulation of mutagenesis in eukaryotes by DNA replication fork dynamics and quality of nucleotide pools

Waisertreiger

Liston

Menezes

et al. 2012

Environ and Mol Mutagen

View full text Add to dashboard Cite

The rate of mutations in eukaryotes depends on a plethora of factors and is not immediately derived from the fidelity of DNA polymerases (Pols). Replication of chromosomes containing the anti-parallel strands of duplex DNA occurs through the copying of leading and lagging strand templates by a trio of Pols α, δ and ε, with the assistance of Pol ζ and Y-family Pols at difficult DNA template structures or sites of DNA damage. The parameters of the synthesis at a given location are dictated by the quality and quantity of nucleotides in the pools, replication fork architecture, transcription status, regulation of Pol switches, and structure of chromatin. The result of these transactions is a subject of survey and editing by DNA repair.

show abstract

Chromosomal Fragmentation Is the Major Consequence of the rdgB Defect in Escherichia coli

Cited by 22 publications

References 16 publications

Synthetic Lethality with the dut Defect in Escherichia coli Reveals Layers of DNA Damage of Increasing Complexity Due to Uracil Incorporation

Synthetic Lethality with the dut Defect in Escherichia coli Reveals Layers of DNA Damage of Increasing Complexity Due to Uracil Incorporation

Hypoxanthine Incorporation Is Nonmutagenic in Escherichia coli

Modulation of mutagenesis in eukaryotes by DNA replication fork dynamics and quality of nucleotide pools

Contact Info

Product

Resources

About