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The key DNA-binding residues in the C-terminal domain of Mycobacterium tuberculosis DNA gyrase A subunit (GyrA).

Huang YY, Deng JY, Gu J, Zhang ZP, Maxwell A, Bi LJ, Chen YY, Zhou YF, Yu ZN, Zhang XE - Nucleic Acids Res. (2006)

Bottom Line: The results show that Y577, R691 and R745 are among the key DNA-binding residues in M.tuberculosis GyrA-CTD, and that the third blade of the GyrA-CTD is the main DNA-binding region in M.tuberculosis DNA gyrase.The substitutions of Y577A, D669A, R691A, R745A and G729W led to the loss of supercoiling and relaxation activities, although they had a little effect on the drug-dependent DNA cleavage and decatenation activities, and had no effect on the ATPase activity.Taken together, these results showed that the GyrA-CTD is essential to DNA gyrase of M.tuberculosis, and promote the idea that the M.tuberculosis GyrA-CTD is a new potential target for drug design.

View Article: PubMed Central - PubMed

Affiliation: State Key Laboratory of Virology, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan 430071, China.

ABSTRACT
As only the type II topoisomerase is capable of introducing negative supercoiling, DNA gyrase is involved in crucial cellular processes. Although the other domains of DNA gyrase are better understood, the mechanism of DNA binding by the C-terminal domain of the DNA gyrase A subunit (GyrA-CTD) is less clear. Here, we investigated the DNA-binding sites in the GyrA-CTD of Mycobacterium tuberculosis gyrase through site-directed mutagenesis. The results show that Y577, R691 and R745 are among the key DNA-binding residues in M.tuberculosis GyrA-CTD, and that the third blade of the GyrA-CTD is the main DNA-binding region in M.tuberculosis DNA gyrase. The substitutions of Y577A, D669A, R691A, R745A and G729W led to the loss of supercoiling and relaxation activities, although they had a little effect on the drug-dependent DNA cleavage and decatenation activities, and had no effect on the ATPase activity. Taken together, these results showed that the GyrA-CTD is essential to DNA gyrase of M.tuberculosis, and promote the idea that the M.tuberculosis GyrA-CTD is a new potential target for drug design. It is the first time that the DNA-binding sites in GyrA-CTD have been identified.

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SPR experiment of DNA-GyrA bindings. (A) Sensorgram of the GyrA and its mutants in absence of GyrB. (B) Sensorgram of the GyrA and its mutants in presence of equal molar concentration (0.1 μM) of GyrB. The inserted table lists the kinetic constants derived from the sensorgrams.
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fig10: SPR experiment of DNA-GyrA bindings. (A) Sensorgram of the GyrA and its mutants in absence of GyrB. (B) Sensorgram of the GyrA and its mutants in presence of equal molar concentration (0.1 μM) of GyrB. The inserted table lists the kinetic constants derived from the sensorgrams.

Mentions: Although the DNA-binding activity of the six mutants was measured by gel-retardation assay, it was not clear whether the mutants were impaired just in wrapping or whether they are unable to bind DNA at all. SPR technology, a sensitive means to measure the molecular interactions, was employed to study interaction between DNA and the GyrA mutants either in the absence or presence of GyrB. The SPR 3000 system contains a dual-channel measuring cell. Both reference and sample channels have the same streptavidin-modified sensor chip, but the chip in the sample channel was modified with a 290 bp dsDNA through streptavidin–biotin interaction. GyrA samples were repeatedly injected in the flow-through carrying buffer. Response signal (response unit, RU) represents the binding of GyrA proteins to the bound DNA. As shown in Figure 10, no response was observed for the mutants Y577A, D691A and R745A with or without GyrB, indicating that these mutants have completely lost the capability of DNA binding. D669A did not bind DNA in the absence of GyrB, but had a weak response signal when GyrB was present, which agrees with the results of the gel-retardation assay (Figure 4). Both E514A and G729W could interact with DNA in the absence or presence of GyrB with significantly lowered binding signal, comparing with the wild-type GyrA or A2B2. The sensorgram shows that the binding mode of the G729W had changed from rapid to slow association and dissociation kinetics.


The key DNA-binding residues in the C-terminal domain of Mycobacterium tuberculosis DNA gyrase A subunit (GyrA).

Huang YY, Deng JY, Gu J, Zhang ZP, Maxwell A, Bi LJ, Chen YY, Zhou YF, Yu ZN, Zhang XE - Nucleic Acids Res. (2006)

SPR experiment of DNA-GyrA bindings. (A) Sensorgram of the GyrA and its mutants in absence of GyrB. (B) Sensorgram of the GyrA and its mutants in presence of equal molar concentration (0.1 μM) of GyrB. The inserted table lists the kinetic constants derived from the sensorgrams.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC1636481&req=5

fig10: SPR experiment of DNA-GyrA bindings. (A) Sensorgram of the GyrA and its mutants in absence of GyrB. (B) Sensorgram of the GyrA and its mutants in presence of equal molar concentration (0.1 μM) of GyrB. The inserted table lists the kinetic constants derived from the sensorgrams.
Mentions: Although the DNA-binding activity of the six mutants was measured by gel-retardation assay, it was not clear whether the mutants were impaired just in wrapping or whether they are unable to bind DNA at all. SPR technology, a sensitive means to measure the molecular interactions, was employed to study interaction between DNA and the GyrA mutants either in the absence or presence of GyrB. The SPR 3000 system contains a dual-channel measuring cell. Both reference and sample channels have the same streptavidin-modified sensor chip, but the chip in the sample channel was modified with a 290 bp dsDNA through streptavidin–biotin interaction. GyrA samples were repeatedly injected in the flow-through carrying buffer. Response signal (response unit, RU) represents the binding of GyrA proteins to the bound DNA. As shown in Figure 10, no response was observed for the mutants Y577A, D691A and R745A with or without GyrB, indicating that these mutants have completely lost the capability of DNA binding. D669A did not bind DNA in the absence of GyrB, but had a weak response signal when GyrB was present, which agrees with the results of the gel-retardation assay (Figure 4). Both E514A and G729W could interact with DNA in the absence or presence of GyrB with significantly lowered binding signal, comparing with the wild-type GyrA or A2B2. The sensorgram shows that the binding mode of the G729W had changed from rapid to slow association and dissociation kinetics.

Bottom Line: The results show that Y577, R691 and R745 are among the key DNA-binding residues in M.tuberculosis GyrA-CTD, and that the third blade of the GyrA-CTD is the main DNA-binding region in M.tuberculosis DNA gyrase.The substitutions of Y577A, D669A, R691A, R745A and G729W led to the loss of supercoiling and relaxation activities, although they had a little effect on the drug-dependent DNA cleavage and decatenation activities, and had no effect on the ATPase activity.Taken together, these results showed that the GyrA-CTD is essential to DNA gyrase of M.tuberculosis, and promote the idea that the M.tuberculosis GyrA-CTD is a new potential target for drug design.

View Article: PubMed Central - PubMed

Affiliation: State Key Laboratory of Virology, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan 430071, China.

ABSTRACT
As only the type II topoisomerase is capable of introducing negative supercoiling, DNA gyrase is involved in crucial cellular processes. Although the other domains of DNA gyrase are better understood, the mechanism of DNA binding by the C-terminal domain of the DNA gyrase A subunit (GyrA-CTD) is less clear. Here, we investigated the DNA-binding sites in the GyrA-CTD of Mycobacterium tuberculosis gyrase through site-directed mutagenesis. The results show that Y577, R691 and R745 are among the key DNA-binding residues in M.tuberculosis GyrA-CTD, and that the third blade of the GyrA-CTD is the main DNA-binding region in M.tuberculosis DNA gyrase. The substitutions of Y577A, D669A, R691A, R745A and G729W led to the loss of supercoiling and relaxation activities, although they had a little effect on the drug-dependent DNA cleavage and decatenation activities, and had no effect on the ATPase activity. Taken together, these results showed that the GyrA-CTD is essential to DNA gyrase of M.tuberculosis, and promote the idea that the M.tuberculosis GyrA-CTD is a new potential target for drug design. It is the first time that the DNA-binding sites in GyrA-CTD have been identified.

Show MeSH
Related in: MedlinePlus