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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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Decatenation activity of the GyrA mutants in the presence of GyrB. GyrA and GyrB were mixed together at an equal molar concentration at 25°C for ∼20 min before the assay. Lane 1, catenated kDNA only; lanes 2–8, catenated kDNA treated with wild-type A2B2, E514A, Y577A, D669A, R691A, G729W and R745A, respectively. Gyrase concentrations were 1 μM in (A), 0.1 μM in (B) and 0.01 μM in (C), respectively.
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fig8: Decatenation activity of the GyrA mutants in the presence of GyrB. GyrA and GyrB were mixed together at an equal molar concentration at 25°C for ∼20 min before the assay. Lane 1, catenated kDNA only; lanes 2–8, catenated kDNA treated with wild-type A2B2, E514A, Y577A, D669A, R691A, G729W and R745A, respectively. Gyrase concentrations were 1 μM in (A), 0.1 μM in (B) and 0.01 μM in (C), respectively.

Mentions: The decatenation activity of the GyrA mutants was tested in the presence of GyrB. As shown in Figure 8A and B, all mutants remained decatenation activity when the gyrase concentrations was 1 μM or 0.1 μM. However, lowering gyrase concentration to 0.01 μM, mutants of D669A, R691A, G729A and R745A showed no decatenation activity, whereas the wild-type enzyme and E514A still had (Figure 8C). The data suggests some facts that decatenation activity of E514A is similar to that of the wild-type, TB gyrase is a strong decatenase (40) and GyrA-CTD is less important for decatenation reaction.


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)

Decatenation activity of the GyrA mutants in the presence of GyrB. GyrA and GyrB were mixed together at an equal molar concentration at 25°C for ∼20 min before the assay. Lane 1, catenated kDNA only; lanes 2–8, catenated kDNA treated with wild-type A2B2, E514A, Y577A, D669A, R691A, G729W and R745A, respectively. Gyrase concentrations were 1 μM in (A), 0.1 μM in (B) and 0.01 μM in (C), respectively.
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Related In: Results  -  Collection

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fig8: Decatenation activity of the GyrA mutants in the presence of GyrB. GyrA and GyrB were mixed together at an equal molar concentration at 25°C for ∼20 min before the assay. Lane 1, catenated kDNA only; lanes 2–8, catenated kDNA treated with wild-type A2B2, E514A, Y577A, D669A, R691A, G729W and R745A, respectively. Gyrase concentrations were 1 μM in (A), 0.1 μM in (B) and 0.01 μM in (C), respectively.
Mentions: The decatenation activity of the GyrA mutants was tested in the presence of GyrB. As shown in Figure 8A and B, all mutants remained decatenation activity when the gyrase concentrations was 1 μM or 0.1 μM. However, lowering gyrase concentration to 0.01 μM, mutants of D669A, R691A, G729A and R745A showed no decatenation activity, whereas the wild-type enzyme and E514A still had (Figure 8C). The data suggests some facts that decatenation activity of E514A is similar to that of the wild-type, TB gyrase is a strong decatenase (40) and GyrA-CTD is less important for decatenation reaction.

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