DNA Replication across α-l-(3′-2′)-Threofuranosyl Nucleotides Mediated by Human DNA Polymerase η

Rachana Tomar, Pratibha P. Ghodke, Amritraj Patra, Elizabeth Smyth, Alexander Pontarelli, William Copp, F. Peter Guengerich, John J. Chaput, Christopher J. Wilds, Michael P. Stone, Martin Egli
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Abstract

α-l-(3′-2′)-Threofuranosyl nucleic acid (TNA) pairs with itself, cross-pairs with DNA and RNA, and shows promise as a tool in synthetic genetics, diagnostics, and oligonucleotide therapeutics. We studied in vitro primer insertion and extension reactions catalyzed by human trans-lesion synthesis (TLS) DNA polymerase η (hPol η) opposite a TNA-modified template strand without and in combination with O4-alkyl thymine lesions. Across TNA-T (tT), hPol η inserted mostly dAMP and dGMP, dTMP and dCMP with lower efficiencies, followed by extension of the primer to a full-length product. hPol η inserted dAMP opposite O4-methyl and -ethyl analogs of tT, albeit with reduced efficiencies relative to tT. Crystal structures of ternary hPol η complexes with template tT and O4-methyl tT at the insertion and extension stages demonstrated that the shorter backbone and different connectivity of TNA compared to DNA (3′ → 2′ versus 5′ → 3′, respectively) result in local differences in sugar orientations, adjacent phosphate spacings, and directions of glycosidic bonds. The 3′-OH of the primer’s terminal thymine was positioned at 3.4 Å on average from the α-phosphate of the incoming dNTP, consistent with insertion opposite and extension past the TNA residue by hPol η. Conversely, the crystal structure of a ternary hPol η·DNA·tTTP complex revealed that the primer’s terminal 3′-OH was too distant from the tTTP α-phosphate, consistent with the inability of the polymerase to incorporate TNA. Overall, our study provides a better understanding of the tolerance of a TLS DNA polymerase vis-à-vis unnatural nucleotides in the template and as the incoming nucleoside triphosphate.

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由人类 DNA 聚合酶 η 介导的跨 α-l-(3′-2′)-三呋喃糖基核苷酸的 DNA 复制
α-l-(3′-2′)-三呋喃糖基核酸(TNA)可与自身配对,也可与 DNA 和 RNA 交叉配对,有望成为合成遗传学、诊断学和寡核苷酸疗法的工具。我们研究了人类反式离子合成(TLS)DNA聚合酶η(hPol η)在TNA修饰的模板链对面催化的体外引物插入和延伸反应。在 TNA-T(tT)对面,hPol η 主要插入 dAMP 和 dGMP,dTMP 和 dCMP 的效率较低,随后引物延伸为全长产物。hPol η 与模板 tT 和 O4-甲基 tT 的三元复合物在插入和延伸阶段的晶体结构表明,与 DNA 相比,TNA 的骨架更短,连接性也不同(分别为 3′ → 2′ 与 5′ → 3′),这导致了糖的取向、相邻磷酸间距和糖苷键方向的局部差异。引物末端胸腺嘧啶的 3′-OH 与进入的 dNTP 的 α-磷酸的平均距离为 3.4 Å,这与 hPol η 在 TNA 残基对面插入并延伸过去的情况一致。相反,hPol η-DNA-tTTP 三元复合物的晶体结构显示,引物末端的 3′-OH与 tTTP α-磷酸的距离太远,这与聚合酶无法结合 TNA 的情况一致。总之,我们的研究让人们更好地了解了 TLS DNA 聚合酶对模板中的非天然核苷酸和输入的三磷酸核苷的耐受性。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
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