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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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GyrA D82G gyrase supports DNA replication less efficiently than the wild-type gyrase. The standard oriC replication reactions contained an oriC plasmid pBROTB535 type I (35 fmol as molecule = 420 pmol nt) as a template and the reaction was incubated at 30°C for 10 min. Incorporations of nt into acid-insoluble products were: 35 (lane 1), 43 (lane 1), 68 (lane 3), 115 (lane 4), 142 (lane 5), 44 (lane 6), 59 (lane 7), 88 (lane 8), 120 (lane 9), 51 (lane 10), 77 (lane 11), 132 (lane 12) and 154 (lane 13) pmol nt. LRI and ERI are late and early replication intermediates, respectively. Abbreviations were as in the legend to Figure 1.
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fig2: GyrA D82G gyrase supports DNA replication less efficiently than the wild-type gyrase. The standard oriC replication reactions contained an oriC plasmid pBROTB535 type I (35 fmol as molecule = 420 pmol nt) as a template and the reaction was incubated at 30°C for 10 min. Incorporations of nt into acid-insoluble products were: 35 (lane 1), 43 (lane 1), 68 (lane 3), 115 (lane 4), 142 (lane 5), 44 (lane 6), 59 (lane 7), 88 (lane 8), 120 (lane 9), 51 (lane 10), 77 (lane 11), 132 (lane 12) and 154 (lane 13) pmol nt. LRI and ERI are late and early replication intermediates, respectively. Abbreviations were as in the legend to Figure 1.

Mentions: Another transcriptional property of a strain carrying the D82G mutation is the constitutive induction of the dinP gene, encoding a Y-family DNA polymerase mediating translesion DNA synthesis (50). Since, despite the compensatory transcriptional changes in the mutant strain, the overall transcriptional state of the cells retained the signature of a suboptimal supercoiling activity, it seemed plausible that the constitutive activation of dinP might be reflecting an elevated steady-state level of DNA damage in the strain. One possible source of such damage could be the DNA replication itself, which may have a higher level of spontaneous arrests than in the wild type when elongation swivel, provided by DNA gyrase, is not fully effective (51). We examined the ability of the mutant gyrase to support DNA replication in vitro (Figure 2). GyrA D82G gyrase could not support oriC DNA replication reaction as efficiently as the wild-type gyrase could. These results indicated that the D82G mutation might affect the swivel activity of gyrase during chromosomal replication, which could elevate the steady-state level of DNA damage.


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)

GyrA D82G gyrase supports DNA replication less efficiently than the wild-type gyrase. The standard oriC replication reactions contained an oriC plasmid pBROTB535 type I (35 fmol as molecule = 420 pmol nt) as a template and the reaction was incubated at 30°C for 10 min. Incorporations of nt into acid-insoluble products were: 35 (lane 1), 43 (lane 1), 68 (lane 3), 115 (lane 4), 142 (lane 5), 44 (lane 6), 59 (lane 7), 88 (lane 8), 120 (lane 9), 51 (lane 10), 77 (lane 11), 132 (lane 12) and 154 (lane 13) pmol nt. LRI and ERI are late and early replication intermediates, respectively. Abbreviations were as in the legend to Figure 1.
© Copyright Policy - openaccess
Related In: Results  -  Collection

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

fig2: GyrA D82G gyrase supports DNA replication less efficiently than the wild-type gyrase. The standard oriC replication reactions contained an oriC plasmid pBROTB535 type I (35 fmol as molecule = 420 pmol nt) as a template and the reaction was incubated at 30°C for 10 min. Incorporations of nt into acid-insoluble products were: 35 (lane 1), 43 (lane 1), 68 (lane 3), 115 (lane 4), 142 (lane 5), 44 (lane 6), 59 (lane 7), 88 (lane 8), 120 (lane 9), 51 (lane 10), 77 (lane 11), 132 (lane 12) and 154 (lane 13) pmol nt. LRI and ERI are late and early replication intermediates, respectively. Abbreviations were as in the legend to Figure 1.
Mentions: Another transcriptional property of a strain carrying the D82G mutation is the constitutive induction of the dinP gene, encoding a Y-family DNA polymerase mediating translesion DNA synthesis (50). Since, despite the compensatory transcriptional changes in the mutant strain, the overall transcriptional state of the cells retained the signature of a suboptimal supercoiling activity, it seemed plausible that the constitutive activation of dinP might be reflecting an elevated steady-state level of DNA damage in the strain. One possible source of such damage could be the DNA replication itself, which may have a higher level of spontaneous arrests than in the wild type when elongation swivel, provided by DNA gyrase, is not fully effective (51). We examined the ability of the mutant gyrase to support DNA replication in vitro (Figure 2). GyrA D82G gyrase could not support oriC DNA replication reaction as efficiently as the wild-type gyrase could. These results indicated that the D82G mutation might affect the swivel activity of gyrase during chromosomal replication, which could elevate the steady-state level of DNA damage.

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