Siberian Branch of the Russian Academy of Sciences Institute of Chemical Biology and Fundamental Medicine, 8 Lavrentieva Ave., 630090 Novosibirsk, Russia.
Department of Natural Sciences, Novosibirsk State University, 2 Pirogova St., 630090 Novosibirsk, Russia.
Int J Mol Sci. 2024 May 30;25(11):6006. doi: 10.3390/ijms25116006.
The DNA building blocks 2'-deoxynucleotides are enantiomeric, with their natural β-D-configuration dictated by the sugar moiety. Their synthetic β-L-enantiomers (βLdNs) can be used to obtain L-DNA, which, when fully substituted, is resistant to nucleases and is finding use in many biosensing and nanotechnology applications. However, much less is known about the enzymatic recognition and processing of individual βLdNs embedded in D-DNA. Here, we address the template properties of βLdNs for several DNA polymerases and the ability of base excision repair enzymes to remove these modifications from DNA. The Klenow fragment was fully blocked by βLdNs, whereas DNA polymerase κ bypassed them in an error-free manner. Phage RB69 DNA polymerase and DNA polymerase β treated βLdNs as non-instructive but the latter enzyme shifted towards error-free incorporation on a gapped DNA substrate. DNA glycosylases and AP endonucleases did not process βLdNs. DNA glycosylases sensitive to the base opposite their cognate lesions also did not recognize βLdNs as a correct pairing partner. Nevertheless, when placed in a reporter plasmid, pyrimidine βLdNs were resistant to repair in human cells, whereas purine βLdNs appear to be partly repaired. Overall, βLdNs are unique modifications that are mostly non-instructive but have dual non-instructive/instructive properties in special cases.
DNA 结构单元 2'-脱氧核苷酸是对映异构体，其天然的β-D-构型由糖部分决定。它们的合成β-L-对映异构体（βLdNs）可用于获得 L-DNA，当完全取代时，L-DNA 对核酸酶具有抗性，并在许多生物传感和纳米技术应用中得到应用。然而，对于嵌入在 D-DNA 中的单个βLdN 的酶促识别和加工，人们知之甚少。在这里，我们研究了几种 DNA 聚合酶对βLdN 的模板性质，以及碱基切除修复酶从 DNA 中去除这些修饰的能力。Klenow 片段被βLdNs 完全阻断，而 DNA 聚合酶 κ 以无错误的方式绕过它们。噬菌体 RB69 DNA 聚合酶和 DNA 聚合酶 β 将βLdNs 视为非指令性底物，但后者酶在缺口 DNA 底物上更倾向于无错误掺入。DNA 糖苷酶和 AP 内切核酸酶不能处理βLdNs。与它们同源损伤相对应的碱基敏感的 DNA 糖苷酶也不能将βLdNs 识别为正确的配对伙伴。然而，当放置在报告质粒中时，嘧啶βLdNs 在人类细胞中不易修复，而嘌呤βLdNs 似乎部分被修复。总体而言，βLdNs 是独特的修饰，大多数情况下是非指令性的，但在特殊情况下具有双重非指令性/指令性性质。
