Deinococcus radiodurans is a bacterium with extreme resistance to desiccation and radiation. Although the origins of this extreme resistance have not been fully elucidated, an efficient DNA repair machinery that includes the enzyme DNA polymerase I, is potentially crucial as part of a protection mechanism.

Here we have cloned and performed small, medium, and large-scale expression of full-length D. radiodurans DNA polymerase I (DrPolI) as well as the large/Klenow fragment (DrKlenow). We then carried out functional characterization of 5′ exonuclease, DNA strand displacement and polymerase activities of these proteins using gel-based and molecular beacon-based biochemical assays. With the same expression and purification strategy, we got higher yield in the production of DrKlenow than of the full-length protein, approximately 2.5 mg per liter of culture. Moreover, we detected a prominent 5′ exonuclease activity of DrPolI in vitro. This activity and, DrKlenow strand displacement and DNA polymerase activities are preferentially stimulated at pH 8.0–8.5 and are reduced by addition of NaCl. Interestingly, both protein variants are more thermostable at pH 6.0–6.5.

The characterization of DrPolI's multiple functions provides new insights into the enzyme's role in DNA repair pathways, and how the modulation of these functions is potentially used by D. radiodurans as a survival strategy.

Deinococcus radiodurans是一种对干燥和辐射具有极强抵抗力的细菌。尽管这种极端抗性的起源尚未完全阐明，但包括 DNA 聚合酶 I 在内的有效 DNA 修复机制作为保护机制的一部分可能是至关重要的。

在这里，我们克隆并进行了全长D. radiodurans DNA 聚合酶 I (DrPolI) 以及大/Klenow 片段 (DrKlenow) 的小、中和大规模表达。然后，我们使用基于凝胶和基于分子信标的生化测定对这些蛋白质的 5' 外切核酸酶、DNA 链置换和聚合酶活性进行了功能表征。使用相同的表达和纯化策略，我们在 DrKlenow 的生产中获得了比全长蛋白质更高的产量，大约每升培养物 2.5 mg。此外，我们在体外检测到 DrPolI 显着的 5' 核酸外切酶活性。这种活性以及 DrKlenow 链置换和 DNA 聚合酶活性在 pH 8.0-8.5 时优先受到刺激，并通过添加 NaCl 降低。有趣的是，这两种蛋白质变体在 pH 6.0–6.5 时都更热稳定。

DrPolI 的多种功能的表征为该酶在 DNA 修复途径中的作用提供了新的见解，以及这些功能的调节如何被D. radiodurans潜在地用作生存策略。