Measurement of Intracellular Magnesium Concentration in 3T3 Fibroblasts with the Fluorescent Indicator Mag-indo-1

Morelle, Bruno; Salmon, Jean‐Marie; Vigo, Jean; Viallet, Pierre

doi:10.1006/abio.1994.1156

Cited by 27 publications

(19 citation statements)

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“…We found that, in the High Mg 2ϩ reactions, MOPS buffers with pH values between 7.0 and 7.5 would substitute for the Tris acetate and that omission of the ATP-regenerating system did not affect the (presumably free Mg 2ϩ ion) is thus the salient feature distinguishing the two reaction conditions, in accord with previous observations (22,24). In the experiments reported below, either 5Ј or internal 32 P labels were used, to allow detection of Chispecific reaction products under either reaction condition.…”

Section: Mgsupporting

confidence: 88%

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Strand Specificity of Nicking of DNA at Chi Sites by RecBCD Enzyme

Taylor

Smith

1995

Journal of Biological Chemistry

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RecBCD enzyme is essential for the major pathway of homologous recombination of linear DNA in Escherichia coli. It is a potent nuclease and helicase and, during its unwinding of double-stranded DNA, makes singlestrand scissions in the vicinity of Chi recombination hot spots. We report here that both the strand that is cut and the position of the cuts relative to Chi depended on the ATP to Mg 2؉ ratio. With ATP in excess, Chi-dependent nicks occurred, as we have previously reported, four to six nucleotides to the 3-side of the Chi octamer (5-GCTGGTGG-3) and were detected only on the strand bearing that sequence. Three differences were seen with Mg 2؉ in excess. 1) Chi-dependent 3-ends were produced on the GCTGGTGG-containing strand closer to and within the Chi octamer. 2) Chi-dependent cuts occurred on the complementary DNA strand. 3) RecBCD enzyme destroyed the 3-terminated strand of DNA from its entry point up to the vicinity of the Chi site, as others have previously reported. We show here that, with Mg 2؉ in excess, the enzyme continued to travel along DNA, after encountering a Chi site, releasing both strands of the DNA distal to Chi as single strands. We discuss potential biological consequences of these two modes of RecBCD enzyme-Chi interaction.RecBCD enzyme (EC 3.1.11.5), encoded by the recB, recC, and recD genes of Escherichia coli, is the best characterized enzyme specific to the major (RecBCD) pathway of homologous recombination in E. coli conjugation and transduction (reviewed in Ref. 1). Purified RecBCD has many activities. It has potent ATP-dependent ss 1 and ds DNA exonuclease activities and a weak ATP-stimulated endonuclease activity on ss, but not ds, circular DNA. Under conditions that reduce these nuclease activities (e.g. 5 mM ATP and 1 mM Mg 2ϩ ) the enzyme totally unwinds ds DNA, up to 40 kilobase pairs or longer in the presence of SSB (2-4). Electron microscopy revealed that the enzyme travels unidirectionally and highly processively from a ds DNA end through the DNA, with either transient or permanent unwinding of the DNA behind the enzyme (5, 6). Under such conditions the enzyme has a potent site-specific cleavage activity at Chi sequences (5Ј-GCTGGTGG-3Ј) (7), a recombinational hot spot specific to the RecBCD pathway (reviewed in Ref. 8). This cleavage occurs only as the enzyme is unwinding the DNA from right to left, as Chi is written here (9). The enzyme makes Chi-dependent ss nicks a few bases to the 3Ј-side of Chi on the GCTGGTGG-containing ("Upper") strand 2 but makes no detectable Chi-dependent nicks on the complementary strand (7, 9) (Fig. 1, left). The activities of the purified enzyme, under these conditions, correlate well with the properties of Chi deduced from genetic studies and support a previously proposed model of Chi-stimulated recombination by the RecBCD pathway (8, 10) (see "Discussion").RecBCD enzyme has been coupled with RecA and SSB proteins to produce Chi-dependent joint molecules from linear Chi-containing ds DNA and supercoiled Chi-free ds DNA (11)(12)(13). This...

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Section: Mgsupporting

confidence: 88%

“…The self-complementary oligonucleotide shown was 5Ј-end labeled with 32 P, and some of it was self-ligated (14). Monomer and dimer length molecules were purified by gel electrophoresis.…”

Section: Fig 3 Resistance Of Hairpin Ends To Recbcd Enzyme Degradatmentioning

confidence: 99%

Strand Specificity of Nicking of DNA at Chi Sites by RecBCD Enzyme

Taylor

Smith

1995

Journal of Biological Chemistry

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“…Using Mag-indo1 (22) and Mag-fura 2 (24) Ca 2ϩ concentration is found to be between 127 and 200 M, and null point measurements (41) yield concentrations in the range of 21-47 M, while the use of aequorin (42) results in a value of 2 mM [the latter to be compared to results with a targeted photoprotein, i.e., 0.3-0.5 M (43-45)]. These very large differences using a variety of methodological approaches might partially be due to the fluorescence of protein-bound probes, such as in reference to protein-bound Magindo1 and Mag-fura 2 (13,19,20 (43)(44)(45) obtained [Ca 2ϩ ] values between 0.3 and 5 M (as compared to 500 nM calculated from the experiments reported here).…”

Section: Discussionmentioning

confidence: 97%

“…The application of the probe can be enhanced by combining: (i) confocal microspectrofluorometry which allows the recording of fluorescence spectra from cell volumes under 1 m 3 (46), and (ii) a resolution method of the cell fluorescence spectrum enabling consideration of the contribution of the protein-bound probe component (19,20).…”

Section: Discussionmentioning

confidence: 99%

“…Figure 5a gives an example of such a spectrum. As already shown by Morelle et al (17,20) this fluorescence spectrum had to be resolved into its components, i.e., proteinbound (LP), cation-bound (LC), and free (L) Mag-indo1 fluorescence spectra. After digitonin permeabilization, we monitored the evolution of cell fluorescence spectra: thus, we obtained the fluorescence intensity of L, LC, and LP components at different times (Figs.…”

Section: Fluorescence Spectrum Analysis Of Mag-indo1 Trapped In Organmentioning

confidence: 94%

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