Malik Shuja S, Coey Christopher T, Varney Kristen M, Pozharski Edwin, Drohat Alexander C
Department of Biochemistry and Molecular Biology, University of Maryland School of Medicine, Baltimore, MD 21201, USA.
Department of Biochemistry and Molecular Biology, University of Maryland School of Medicine, Baltimore, MD 21201, USA University of Maryland Marlene and Stewart Greenebaum Cancer Center, Baltimore, MD 21201, USA Center for Biomolecular Therapeutics, Institute for Bioscience and Biotechnology Research, Rockville, MD 20850, USA.
Nucleic Acids Res. 2015 Oct 30;43(19):9541-52. doi: 10.1093/nar/gkv890. Epub 2015 Sep 10.
Thymine DNA Glycosylase (TDG) performs essential functions in maintaining genetic integrity and epigenetic regulation. Initiating base excision repair, TDG removes thymine from mutagenic G ·: T mispairs caused by 5-methylcytosine (mC) deamination and other lesions including uracil (U) and 5-hydroxymethyluracil (hmU). In DNA demethylation, TDG excises 5-formylcytosine (fC) and 5-carboxylcytosine (caC), which are generated from mC by Tet (ten-eleven translocation) enzymes. Using improved crystallization conditions, we solved high-resolution (up to 1.45 Å) structures of TDG enzyme-product complexes generated from substrates including G·U, G·T, G·hmU, G·fC and G·caC. The structures reveal many new features, including key water-mediated enzyme-substrate interactions. Together with nuclear magnetic resonance experiments, the structures demonstrate that TDG releases the excised base from its tight product complex with abasic DNA, contrary to previous reports. Moreover, DNA-free TDG exhibits no significant binding to free nucleobases (U, T, hmU), indicating a Kd >> 10 mM. The structures reveal a solvent-filled channel to the active site, which might facilitate dissociation of the excised base and enable caC excision, which involves solvent-mediated acid catalysis. Dissociation of the excised base allows TDG to bind the beta rather than the alpha anomer of the abasic sugar, which might stabilize the enzyme-product complex.
胸腺嘧啶DNA糖基化酶（TDG）在维持基因完整性和表观遗传调控中发挥着重要作用。TDG启动碱基切除修复，从由5-甲基胞嘧啶（mC）脱氨基导致的诱变G·:T错配以及包括尿嘧啶（U）和5-羟甲基尿嘧啶（hmU）在内的其他损伤中去除胸腺嘧啶。在DNA去甲基化过程中，TDG切除由Tet（十一-易位）酶从mC产生的5-甲酰基胞嘧啶（fC）和5-羧基胞嘧啶（caC）。通过改进结晶条件，我们解析了由包括G·U、G·T、G·hmU、G·fC和G·caC在内的底物生成的TDG酶-产物复合物的高分辨率（高达1.45 Å）结构。这些结构揭示了许多新特征，包括关键的水介导的酶-底物相互作用。结合核磁共振实验，这些结构表明TDG从其与无碱基DNA的紧密产物复合物中释放出切除的碱基，这与先前的报道相反。此外，无DNA的TDG对游离核碱基（U、T、hmU）没有明显的结合，表明解离常数Kd >> 10 mM。这些结构揭示了一个通向活性位点的充满溶剂的通道，这可能有助于切除碱基的解离并实现caC切除，caC切除涉及溶剂介导的酸催化。切除碱基的解离使TDG能够结合无碱基糖的β型而非α型异头物，这可能稳定酶-产物复合物。
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