Howard Hughes Medical Institute, Department of Molecular Biology, and Center for Computational and Integrative Biology, Massachusetts General Hospital, Boston, MA 02114, USA.
Nucleic Acids Res. 2019 Sep 26;47(17):8941-8949. doi: 10.1093/nar/gkz707.
Genetic polymers that could plausibly govern life in the universe might inhabit a broad swath of chemical space. A subset of these genetic systems can exchange information with RNA and DNA and could therefore form the basis for model protocells in the laboratory. N3'→P5' phosphoramidate (NP) DNA is defined by a conservative linkage substitution and has shown promise as a protocellular genetic material, but much remains unknown about its functionality and fidelity due to limited enzymatic tools. Conveniently, we find widespread NP-DNA-dependent DNA polymerase activity among reverse transcriptases, an observation consistent with structural studies of the RNA-like conformation of NP-DNA duplexes. Here, we analyze the consequences of this unnatural template linkage on the kinetics and fidelity of DNA polymerization activity catalyzed by wild-type and variant reverse transcriptases. Template-associated deficits in kinetics and fidelity suggest that even highly conservative template modifications give rise to error-prone DNA polymerase activity. Enzymatic copying of NP-DNA sequences is nevertheless an important step toward the future study and engineering of this synthetic genetic polymer.
可能控制宇宙中生命的遗传聚合物可能存在于广泛的化学空间中。这些遗传系统的一个子集可以与 RNA 和 DNA 交换信息，因此可以作为实验室中模型原细胞的基础。N3'→P5' 磷酰胺（NP）DNA 的定义是保守的键取代，并且由于酶工具有限，作为原细胞遗传物质具有很大的潜力，但由于其功能和保真度仍知之甚少。我们发现，逆转录酶中广泛存在 NP-DNA 依赖性 DNA 聚合酶活性，这一观察结果与 NP-DNA 双链体类似 RNA 构象的结构研究一致。在这里，我们分析了这种非天然模板键对野生型和变体逆转录酶催化的 DNA 聚合酶活性的动力学和保真度的影响。模板相关的动力学和保真度缺陷表明，即使是高度保守的模板修饰也会导致易错的 DNA 聚合酶活性。然而，NP-DNA 序列的酶复制是朝着对这种合成遗传聚合物进行未来研究和工程的重要一步。
