Complexes of the uracil-DNA glycosylase inhibitor protein, Ugi, with Mycobacterium smegmatis and Mycobacterium tuberculosis uracil-DNA glycosylases
Complexes of the uracil-DNA glycosylase inhibitor protein, Ugi, with Mycobacterium smegmatis and Mycobacterium tuberculosis uracil-DNA glycosylases
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DOI：
10.1099/mic.0.26228-0
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发表时间：
2003-07-01
期刊：
影响因子：
2.8
通讯作者：
Varshney, U
中科院分区：
文献类型：
--
作者：
Acharya, N;Kumar, P;Varshney, U
尿嘧啶是一种致突变碱基，通过胞嘧啶脱氨基或DNA聚合酶掺入DUMP而出现在DNA中。DNA中的这种非常规碱基被尿嘧啶-DNA糖基酶(UDG)去除。有趣的是，一种噬菌体编码的短多肽UDG抑制物(UDG Inhibitor，UDG Inhibitor)通过形成紧密的复合体来特异性地抑制UDG。UGI与来自大肠杆菌、人类和单纯疱疹病毒的UDG的复合体的三维结构表明，UGI中的两个结构元件--疏水口袋和β1-边与UDG建立了关键的相互作用。在这份报告中，描述了UGI与来自致病菌结核分枝杆菌和污垢分枝杆菌的UDGs的络合物的表征，耻垢分枝杆菌是前者的模式生物。与大肠杆菌(Eco)UDG-UGI复合体不同，UDG-UGI复合体在8M尿素处理下稳定，而分枝杆菌UDG-UGI复合体在5-6M尿素中解离。此外，分枝杆菌UDG复合体中的UGI可以被DNA底物交换。有趣的是，虽然EcoUDG在与分枝杆菌UDG-UGI复合体孵育时将UGI隔离到EcoUDG-UGI复合体中，但即使大量过剩的分枝杆菌UDG也不能将UGI从EcoUDG-UGI复合体中隔离出来。然而，当MtuUDG与耻垢分枝杆菌(MSM)UDG-UGI或EcoUDG(L191G)-UGI复合体孵育时，可见结核分枝杆菌(MTU)UDG-UGI复合体。UGI与分枝杆菌UDG(天然缺乏与UGI的β1边缘相互作用的一些重要结构元素)和EcoUDG的突变体(缺乏与UGI的疏水口袋相互作用)的可逆性突出了这两类相互作用在形成UDG-UGI复合体中的重要性。此外，研究表明，即使分枝杆菌UDG-UGI复合体在5-6M尿素中解离，UGI仍然是分枝杆菌UDG活性的有效抑制剂。
Uracil, a promutagenic base, appears in DNA either by deamination of cytosine or by incorporation of dUMP by DNA polymerases. This unconventional base in DNA is removed by uracil-DNA glycosylase (UDG). Interestingly, a bacteriophage-encoded short polypeptide, UDG inhibitor (Ugi), specifically inhibits UDGs by forming a tight complex. Three-dimensional structures of the complexes of Ugi with UDGs from Escherichia coli, human and herpes simplex virus have shown that two of the structural elements in Ugi, the hydrophobic pocket and the beta1-edge, establish key interactions with UDGs. In this report the characterization of complexes of Ugi with UDGs from Mycobacterium tuberculosis, a pathogenic bacterium, and Mycobacterium smegmatis, a widely used model organism for the former, is described. Unlike the E coli (Eco) UDG-Ugi complex, which is stable to treatment with 8 M urea, the mycobacterial UDG-Ugi complexes dissociate in 5-6 M urea. Furthermore, the Ugi from the complexes of mycobacterial UDGs can be exchanged by the DNA substrate. Interestingly, while EcoUDG sequestered Ugi into the EcoUDG-Ugi complex when incubated with mycobacterial UDG-Ugi complexes, even a large excess of mycobacterial UDGs failed to sequester Ugi from the EcoUDG-Ugi complex. However, the M. tuberculosis (Mtu) UDG-Ugi complex was seen when MtuUDG was incubated with M. smegmatis (Msm) UDG-Ugi or EcoUDG(L191G)-Ugi complexes. The reversible nature of the complexes of Ugi with mycobacterial UDGs (which naturally lack some of the structural elements important for interaction with the beta1-edge of Ugi) and with mutants of EcoUDG (which are deficient in interaction with the hydrophobic pocket of Ugi) highlights the significance of both classes of interaction in formation of UDG-Ugi complexes. Furthermore, it is shown that even though mycobacterial UDG-Ugi complexes dissociate in 5-6 M urea, Ugi is still a potent inhibitor of UDG activity in mycobacteria.