Defining the impact of sumoylation on substrate binding and catalysis by thymine DNA glycosylase.
Defining the impact of sumoylation on substrate binding and catalysis by thymine DNA glycosylase.
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DOI：
10.1093/nar/gky278
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发表时间：
2018-06-01
影响因子：
14.9
通讯作者：
Drohat AC
中科院分区：
文献类型：
--
作者：
Coey CT;Drohat AC
胸腺嘧啶DNA糖基化酶（TDG）在DNA去甲基化的多步骤途径中，从5-甲基胞嘧啶（mC）脱氨产生的致突变性G·T错对中去除胸腺嘧啶，并去除两个mC衍生物，5-甲酰基胞嘧啶（fC）和5-羧基胞嘧啶（caC）。TDG被小泛素样修饰物（SUMO）蛋白修饰，但SUMO化对TDG活性的影响尚不明确，TDG SUMO化的功能仍不清楚。我们通过SUMO-1或SUMO-2测定了TDG的SUMO-1化对底物结合和催化参数的影响。单次转化实验表明，sumoylation显著降低TDG碱基切除活性，G·T活性降低≥45倍，fC和caC被缓慢切除，反应半衰期≥9 min（37°C）。荧光各向异性研究表明，未经修饰的TDG与G·fC和G·caC底物紧密结合，解离常数在低纳摩尔范围内。虽然TDG的sumoylation削弱了底物结合，但其剩余亲和力是可观的，与生物化学特征的fC和caC读卡器相当。我们的研究结果提出了一种可能性，即sumoylation使TDG至少暂时地起着读取fC和caC的作用。值得注意的是，summoylation可能潜在地促进TDG招募其他蛋白质，包括转录因子或表观遗传调节因子，到DNA中的这些位点。
Thymine DNA glycosylase (TDG) excises thymine from mutagenic G·T mispairs generated by deamination of 5-methylcytosine (mC) and it removes two mC derivatives, 5−formylcytosine (fC) and 5−carboxylcytosine (caC), in a multistep pathway for DNA demethylation. TDG is modified by small ubiquitin-like modifier (SUMO) proteins, but the impact of sumoylation on TDG activity is poorly defined and the functions of TDG sumoylation remain unclear. We determined the effect of TDG sumoylation, by SUMO-1 or SUMO-2, on substrate binding and catalytic parameters. Single turnover experiments reveal that sumoylation dramatically impairs TDG base-excision activity, such that G·T activity is reduced by ≥45-fold and fC and caC are excised slowly, with a reaction half-life of ≥9 min (37°C). Fluorescence anisotropy studies reveal that unmodified TDG binds tightly to G·fC and G·caC substrates, with dissociation constants in the low nanomolar range. While sumoylation of TDG weakens substrate binding, the residual affinity is substantial and is comparable to that of biochemically-characterized readers of fC and caC. Our findings raise the possibility that sumoylation enables TDG to function, at least transiently, as reader of fC and caC. Notably, sumoylation could potentially facilitate TDG recruitment of other proteins, including transcription factors or epigenetic regulators, to these sites in DNA.
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