Dependence of substrate binding and catalysis on pH, ionic strength, and temperature for thymine DNA glycosylase: Insights into recognition and processing of G·T mispairs.
Dependence of substrate binding and catalysis on pH, ionic strength, and temperature for thymine DNA glycosylase: Insights into recognition and processing of G·T mispairs.
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DOI：
10.1016/j.dnarep.2011.03.004
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发表时间：
2011-05-05
期刊：
影响因子：
3.8
通讯作者：
Drohat AC
中科院分区：
文献类型：
--
作者：
Maiti A;Drohat AC
由5-甲基胞嘧啶（m5 C）脱氨基作用引起的G·T错配的修复包括切除胸腺嘧啶和通过碱基切除修复（BER）恢复G·C对。胸腺嘧啶DNA糖基化酶（TDG）是两种能特异性去除G·T错配胸腺嘧啶的哺乳动物酶之一。虽然TDG可以切除其他碱基，但它对CpG背景保持严格的特异性，表明脱氨基的m5 C是重要的生物底物。最近的研究表明，TDG是胚胎发生所必需的;它有助于维持活性染色质复合物并启动BER以对抗异常的从头CpG甲基化，这可能涉及切除活性脱氨基的m5 C。TDG相对较弱的G·T活性与CpG位点的超突变性有关，这主要涉及m5 C脱氨基引起的C→T转换。因此，重要的是要了解TDG如何识别和处理底物，特别是G·T错配。在这里，我们通过研究底物结合和催化对pH值，离子强度和温度的依赖性来扩展我们对TDG的详细研究。催化活性在pH 5.5-9范围内相对恒定，但在pH >9时，由于底物结合严重减弱以及目标碱基潜在电离，催化活性急剧下降，但在pH >9时，催化活性急剧下降，这是因为底物结合严重减弱以及目标碱基潜在电离。福尔斯。随着离子强度的增加，底物结合和催化作用急剧下降，特别是对于G·T底物，部分原因是对核苷酸翻转的影响。TDG在37 °C下快速且不可逆地聚集，但可以通过特异性和非特异性DNA稳定。催化的温度依赖性揭示了G·U和G·T底物的巨大和意想不到的差异，其中G·T活性表现出更陡峭的温度依赖性。结果表明，可逆的核苷酸翻转是更迅速的G·T底物，与我们以前的研究结果一致，空间效应限制了活性位点的寿命的胸腺嘧啶，这可能是相对较弱的G·T活性。我们的研究结果提供了重要的洞察催化TDG，特别是对致突变G·T错配。
Repair of G·T mismatches arising from deamination of 5-methylcytosine (m5C) involves excision of thymine and restoration of a G·C pair via base excision repair (BER). Thymine DNA glycosylase (TDG) is one of two mammalian enzymes that can specifically remove thymine from G·T mispairs. While TDG can excise other bases, it maintains stringent specificity for a CpG context, suggesting deaminated m5C is an important biological substrate. Recent studies reveal TDG is essential for embryogenesis; it helps maintain an active chromatin complex and initiates BER to counter aberrant de novo CpG methylation, which may involve excision of actively deaminated m5C. The relatively weak G·T activity of TDG has been implicated in the hypermutability of CpG sites, which largely involves C→T transitions arising from m5C deamination. Thus, it is important to understand how TDG recognizes and process substrates, particularly G·T mispairs. Here, we extend our detailed studies of TDG by examining the dependence of substrate binding and catalysis on pH, ionic strength, and temperature. Catalytic activity is relatively constant for pH 5.5-9, but falls sharply for pH >9 due to severely weakened substrate binding, and, potentially, ionization of the target base. Substrate binding and catalysis diminish sharply with increasing ionic strength, particularly for G·T substrates, due partly to effects on nucleotide flipping. TDG aggregates rapidly and irreversibly at 37 °C, but can be stabilized by specific and nonspecific DNA. The temperature dependence of catalysis reveals large and unexpected differences for G·U and G·T substrates, where G·T activity exhibits much steeper temperature dependence. The results suggest that reversible nucleotide flipping is much more rapid for G·T substrates, consistent with our previous findings that steric effects limit the active-site lifetime of thymine, which may account for the relatively weak G·T activity. Our findings provide important insight into catalysis by TDG, particularly for mutagenic G·T mispairs.
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