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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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The cleavage activity of GyrA mutants in the presence of GyrB. GyrA and GyrB were mixed together at an equal molar concentration (1.0 μM) at 25°C for ∼20 min before the assay. The concentration of norfloxacin was 60 μg/ml in the reaction buffer. Lane 1, supercoiled pBR322 only; lanes 2–8 are supercoiled pBR322 treated with wild-type A2B2, E514A, Y577A, D669A, R691A, G729W and R745A, respectively, in (A), (B), (C) and (D). The reaction time was (A) 5 min, (B) 10 min, (C) 15 min and (D) 30 min, respectively.
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fig7: The cleavage activity of GyrA mutants in the presence of GyrB. GyrA and GyrB were mixed together at an equal molar concentration (1.0 μM) at 25°C for ∼20 min before the assay. The concentration of norfloxacin was 60 μg/ml in the reaction buffer. Lane 1, supercoiled pBR322 only; lanes 2–8 are supercoiled pBR322 treated with wild-type A2B2, E514A, Y577A, D669A, R691A, G729W and R745A, respectively, in (A), (B), (C) and (D). The reaction time was (A) 5 min, (B) 10 min, (C) 15 min and (D) 30 min, respectively.

Mentions: The drug dependent DNA cleavage activity of the GyrA mutants was tested by incubating the GyrA mutants with norfloxacin. As shown in Figure 7, the drug cleavage activity of E514A was similar to the wild-type A2B2, those of Y577A and G729A were nearly similar to that of the wild-type, and those of D669A, R691A and R745A are less than that of the wild-type. In the presence of GyrB, mutants D669A, R691A and R745A could still cleave DNA completely by increasing reaction time. This suggests that the GyrA-CTD is less important for drug-dependent cleavage activity.


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)

The cleavage activity of GyrA mutants in the presence of GyrB. GyrA and GyrB were mixed together at an equal molar concentration (1.0 μM) at 25°C for ∼20 min before the assay. The concentration of norfloxacin was 60 μg/ml in the reaction buffer. Lane 1, supercoiled pBR322 only; lanes 2–8 are supercoiled pBR322 treated with wild-type A2B2, E514A, Y577A, D669A, R691A, G729W and R745A, respectively, in (A), (B), (C) and (D). The reaction time was (A) 5 min, (B) 10 min, (C) 15 min and (D) 30 min, respectively.
© Copyright Policy
Related In: Results  -  Collection

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

fig7: The cleavage activity of GyrA mutants in the presence of GyrB. GyrA and GyrB were mixed together at an equal molar concentration (1.0 μM) at 25°C for ∼20 min before the assay. The concentration of norfloxacin was 60 μg/ml in the reaction buffer. Lane 1, supercoiled pBR322 only; lanes 2–8 are supercoiled pBR322 treated with wild-type A2B2, E514A, Y577A, D669A, R691A, G729W and R745A, respectively, in (A), (B), (C) and (D). The reaction time was (A) 5 min, (B) 10 min, (C) 15 min and (D) 30 min, respectively.
Mentions: The drug dependent DNA cleavage activity of the GyrA mutants was tested by incubating the GyrA mutants with norfloxacin. As shown in Figure 7, the drug cleavage activity of E514A was similar to the wild-type A2B2, those of Y577A and G729A were nearly similar to that of the wild-type, and those of D669A, R691A and R745A are less than that of the wild-type. In the presence of GyrB, mutants D669A, R691A and R745A could still cleave DNA completely by increasing reaction time. This suggests that the GyrA-CTD is less important for drug-dependent cleavage activity.

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