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RecA can stimulate the relaxation activity of topoisomerase I: Molecular basis of topoisomerase-mediated genome-wide transcriptional responses in Escherichia coli.

Reckinger AR, Jeong KS, Khodursky AB, Hiasa H - Nucleic Acids Res. (2006)

Bottom Line: GyrA D82G gyrase exhibits a reduced supercoiling activity.RecA has no effect on the supercoiling activity of gyrase but stimulates the relaxation activity of topoisomerase I.These results suggest that the functional interaction between RecA and topoisomerase I is responsible for RecA-mediated modulation of the relaxation-dependent transcriptional activity of the Escherichia coli chromosome.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology, University of Minnesota Medical School-Twin Cities, Minneapolis, MN 55455, USA.

ABSTRACT
The superhelicity of the chromosome, which is controlled by DNA topoisomerases, modulates global gene expression. Investigations of transcriptional responses to the modulation of gyrase function have identified two types of topoisomerase-mediated transcriptional responses: (i) steady-state changes elicited by a mutation in gyrase, such as the D82G mutation in GyrA, and (ii) dynamic changes elicited by the inhibition of gyrase. We hypothesize that the steady-state effects are due to the changes in biochemical properties of gyrase, whereas the dynamic effects are due to an imbalance between supercoiling and relaxation activities, which appears to be influenced by the RecA activity. Herein, we present biochemical evidence for hypothesized mechanisms. GyrA D82G gyrase exhibits a reduced supercoiling activity. The RecA protein can influence the balance between supercoiling and relaxation activities either by interfering with the activity of DNA gyrase or by facilitating the relaxation reaction. RecA has no effect on the supercoiling activity of gyrase but stimulates the relaxation activity of topoisomerase I. This stimulation is specific and requires formation of an active RecA filament. These results suggest that the functional interaction between RecA and topoisomerase I is responsible for RecA-mediated modulation of the relaxation-dependent transcriptional activity of the Escherichia coli chromosome.

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RecA stimulates Topo I-catalyzed relaxation reaction. (A) The standard relaxation reaction mixtures containing 100 fmol (as molecule) pBR322 form I DNA and the indicated amounts of Topo I were incubated in the absence (−) or presence (+) of 5.6 μg (3 nt per RecA monomer) of RecA and the DNA products were analyzed as described in Materials and Methods. The assay was repeated three times and virtually identical results were obtained. Representative results are shown. (B) The standard relaxation reaction mixtures containing 100 fmol (as molecule) pBR322 form I DNA and either 0 fmol (−) or 33 fmol (+) of Topo I were incubated in the presence of the indicated amounts of RecA and the DNA products were analyzed as described in Materials and Methods. Two independent experiments showed essentially identical results and representative results are shown.
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fig4: RecA stimulates Topo I-catalyzed relaxation reaction. (A) The standard relaxation reaction mixtures containing 100 fmol (as molecule) pBR322 form I DNA and the indicated amounts of Topo I were incubated in the absence (−) or presence (+) of 5.6 μg (3 nt per RecA monomer) of RecA and the DNA products were analyzed as described in Materials and Methods. The assay was repeated three times and virtually identical results were obtained. Representative results are shown. (B) The standard relaxation reaction mixtures containing 100 fmol (as molecule) pBR322 form I DNA and either 0 fmol (−) or 33 fmol (+) of Topo I were incubated in the presence of the indicated amounts of RecA and the DNA products were analyzed as described in Materials and Methods. Two independent experiments showed essentially identical results and representative results are shown.

Mentions: Next, we examined a possibility that RecA could facilitate the relaxation activity of Topo I. As shown in Figure 4, RecA was capable of stimulating the relaxation activity of Topo I. Based on the amount of Topo I required to completely relax 100 fmol of form I DNA, the relaxation activity of Topo I in the presence of RecA was about 10-fold higher than that in the absence of RecA (Figure 4A). The maximum level of the stimulation of the relaxation activity of Topo I by RecA required a saturated amount (3 nt per RecA monomer) of RecA (Figure 4B). Addition of higher amounts (up to 1 nt per RecA monomer) of RecA did not increase the level of stimulation any further (data not shown). These results suggested that RecA could modulate the balance, or imbalance, between supercoiling and relaxation activities in the cell by directly facilitating the relaxation activity of Topo I.


RecA can stimulate the relaxation activity of topoisomerase I: Molecular basis of topoisomerase-mediated genome-wide transcriptional responses in Escherichia coli.

Reckinger AR, Jeong KS, Khodursky AB, Hiasa H - Nucleic Acids Res. (2006)

RecA stimulates Topo I-catalyzed relaxation reaction. (A) The standard relaxation reaction mixtures containing 100 fmol (as molecule) pBR322 form I DNA and the indicated amounts of Topo I were incubated in the absence (−) or presence (+) of 5.6 μg (3 nt per RecA monomer) of RecA and the DNA products were analyzed as described in Materials and Methods. The assay was repeated three times and virtually identical results were obtained. Representative results are shown. (B) The standard relaxation reaction mixtures containing 100 fmol (as molecule) pBR322 form I DNA and either 0 fmol (−) or 33 fmol (+) of Topo I were incubated in the presence of the indicated amounts of RecA and the DNA products were analyzed as described in Materials and Methods. Two independent experiments showed essentially identical results and representative results are shown.
© Copyright Policy - openaccess
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC1761438&req=5

fig4: RecA stimulates Topo I-catalyzed relaxation reaction. (A) The standard relaxation reaction mixtures containing 100 fmol (as molecule) pBR322 form I DNA and the indicated amounts of Topo I were incubated in the absence (−) or presence (+) of 5.6 μg (3 nt per RecA monomer) of RecA and the DNA products were analyzed as described in Materials and Methods. The assay was repeated three times and virtually identical results were obtained. Representative results are shown. (B) The standard relaxation reaction mixtures containing 100 fmol (as molecule) pBR322 form I DNA and either 0 fmol (−) or 33 fmol (+) of Topo I were incubated in the presence of the indicated amounts of RecA and the DNA products were analyzed as described in Materials and Methods. Two independent experiments showed essentially identical results and representative results are shown.
Mentions: Next, we examined a possibility that RecA could facilitate the relaxation activity of Topo I. As shown in Figure 4, RecA was capable of stimulating the relaxation activity of Topo I. Based on the amount of Topo I required to completely relax 100 fmol of form I DNA, the relaxation activity of Topo I in the presence of RecA was about 10-fold higher than that in the absence of RecA (Figure 4A). The maximum level of the stimulation of the relaxation activity of Topo I by RecA required a saturated amount (3 nt per RecA monomer) of RecA (Figure 4B). Addition of higher amounts (up to 1 nt per RecA monomer) of RecA did not increase the level of stimulation any further (data not shown). These results suggested that RecA could modulate the balance, or imbalance, between supercoiling and relaxation activities in the cell by directly facilitating the relaxation activity of Topo I.

Bottom Line: GyrA D82G gyrase exhibits a reduced supercoiling activity.RecA has no effect on the supercoiling activity of gyrase but stimulates the relaxation activity of topoisomerase I.These results suggest that the functional interaction between RecA and topoisomerase I is responsible for RecA-mediated modulation of the relaxation-dependent transcriptional activity of the Escherichia coli chromosome.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology, University of Minnesota Medical School-Twin Cities, Minneapolis, MN 55455, USA.

ABSTRACT
The superhelicity of the chromosome, which is controlled by DNA topoisomerases, modulates global gene expression. Investigations of transcriptional responses to the modulation of gyrase function have identified two types of topoisomerase-mediated transcriptional responses: (i) steady-state changes elicited by a mutation in gyrase, such as the D82G mutation in GyrA, and (ii) dynamic changes elicited by the inhibition of gyrase. We hypothesize that the steady-state effects are due to the changes in biochemical properties of gyrase, whereas the dynamic effects are due to an imbalance between supercoiling and relaxation activities, which appears to be influenced by the RecA activity. Herein, we present biochemical evidence for hypothesized mechanisms. GyrA D82G gyrase exhibits a reduced supercoiling activity. The RecA protein can influence the balance between supercoiling and relaxation activities either by interfering with the activity of DNA gyrase or by facilitating the relaxation reaction. RecA has no effect on the supercoiling activity of gyrase but stimulates the relaxation activity of topoisomerase I. This stimulation is specific and requires formation of an active RecA filament. These results suggest that the functional interaction between RecA and topoisomerase I is responsible for RecA-mediated modulation of the relaxation-dependent transcriptional activity of the Escherichia coli chromosome.

Show MeSH
Related in: MedlinePlus