The key DNA-binding residues in the C-terminal domain of Mycobacterium tuberculosis DNA gyrase A subunit (GyrA).
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.
Huang
You-Yi
YY
State Key Laboratory of Virology, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan 430071, China.
Deng
Jiao-Yu
JY
Gu
Jing
J
Zhang
Zhi-Ping
ZP
Maxwell
Anthony
A
Bi
Li-Jun
LJ
Chen
Yuan-Yuan
YY
Zhou
Ya-Feng
YF
Yu
Zi-Niu
ZN
Zhang
Xian-En
XE
D000251:Adenosine Triphosphatases / Q000235:genetics / Q000378:metabolism; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D001665:Binding Sites; D027081:DNA Gyrase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D043864:DNA, Catenated / Q000378:metabolism; D004278:DNA, Superhelical / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009169:Mycobacterium tuberculosis / Q000201:enzymology*; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D021122:Protein Subunits / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid
D043864:DNA, Catenated; D004278:DNA, Superhelical; D004268:DNA-Binding Proteins; D021122:Protein Subunits; D000251:Adenosine Triphosphatases; D027081:DNA Gyrase