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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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Related in: MedlinePlus

Relaxation activity of the 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 prior to the assay. Lane 1, supercoiled pBR322 only; lanes 2–8, supercoiled pBR322 treated with wild-type A2B2, E514A, Y577A, D669A, R691A, G729W and R745A, respectively.
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fig6: Relaxation activity of the 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 prior to the assay. Lane 1, supercoiled pBR322 only; lanes 2–8, supercoiled pBR322 treated with wild-type A2B2, E514A, Y577A, D669A, R691A, G729W and R745A, respectively.

Mentions: In the presence of GyrB, all mutants showed nearly no relaxation activity (Figure 6) with the exception of E514A and G729W, but the relaxation activity of E514A was less than that of the wild-type and that of G729W was weak. The results show that residues Y577, D669, R691 and R745 are also involved in the process of relaxing supercoiled DNA, whereas E514 had some role in relaxing DNA. G729W retains DNA-binding activity, but shows very little relaxation 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)

Relaxation activity of the 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 prior to the assay. Lane 1, supercoiled pBR322 only; lanes 2–8, supercoiled pBR322 treated with wild-type A2B2, E514A, Y577A, D669A, R691A, G729W and R745A, respectively.
© Copyright Policy
Related In: Results  -  Collection

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

fig6: Relaxation activity of the 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 prior to the assay. Lane 1, supercoiled pBR322 only; lanes 2–8, supercoiled pBR322 treated with wild-type A2B2, E514A, Y577A, D669A, R691A, G729W and R745A, respectively.
Mentions: In the presence of GyrB, all mutants showed nearly no relaxation activity (Figure 6) with the exception of E514A and G729W, but the relaxation activity of E514A was less than that of the wild-type and that of G729W was weak. The results show that residues Y577, D669, R691 and R745 are also involved in the process of relaxing supercoiled DNA, whereas E514 had some role in relaxing DNA. G729W retains DNA-binding activity, but shows very little relaxation 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