DNA2 is an essential enzyme involved in DNA replication and repair in eukaryotes. In a search for homologues of this protein, we identified and characterised Geobacillus stearothermophilus Bad, a bacterial DNA helicase–nuclease with similarity to human DNA2. We show that Bad contains an Fe-S cluster and identify four cysteine residues that are likely to co-ordinate the cluster by analogy to DNA2. The purified enzyme specifically recognises ss-dsDNA junctions and possesses ssDNA-dependent ATPase, ssDNA binding, ssDNA endonuclease, 5′ to 3′ ssDNA translocase and 5′ to 3′ helicase activity. Single molecule analysis reveals that Bad is a processive DNA motor capable of moving along DNA for distances of >4 kb at a rate of ∼200 bp per second at room temperature. Interestingly, as reported for the homologous human and yeast DNA2 proteins, the DNA unwinding activity of Bad is cryptic and can be unmasked by inactivating the intrinsic nuclease activity. Strikingly, our experiments show that the enzyme loops DNA while translocating, which is an emerging feature of processive DNA unwinding enzymes. The bacterial Bad enzymes will provide an excellent model system for understanding the biochemical properties of DNA2-like helicase–nucleases and DNA looping motor proteins in general.

中文翻译：

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细菌 DNA2 样解旋酶核酸酶的批量和单分子分析揭示了单链 DNA 循环马达。

DNA2 是参与真核生物 DNA 复制和修复的必需酶。为了寻找该蛋白质的同源物，我们鉴定并表征了嗜热脂肪地芽孢杆菌Bad，一种与人类 DNA2 相似的细菌 DNA 解旋酶 - 核酸酶。我们展示了 Bad 包含一个 Fe-S 簇并确定了四个半胱氨酸残基，它们可能通过与 DNA2 类比来协调该簇。纯化的酶特异性识别 ss-dsDNA 连接，并具有 ssDNA 依赖性 ATP 酶、ssDNA 结合、ssDNA 核酸内切酶、5' 到 3' ssDNA 转位酶和 5' 到 3' 解旋酶活性。单分子分析表明，Bad 是一种进行性 DNA 马达，能够在室温下以每秒 200 bp 的速度沿 DNA 移动 >4 kb 的距离。有趣的是，正如对同源人和酵母 DNA2 蛋白所报道的那样，Bad 的 DNA 解旋活性是神秘的，可以通过使内在核酸酶活性失活来揭开。引人注目的是，我们的实验表明，酶在易位时使 DNA 成环，这是进行性 DNA 解旋酶的一个新兴特征。细菌 Bad 酶将为理解 DNA2 样解旋酶 - 核酸酶和 DNA 循环马达蛋白的生化特性提供一个极好的模型系统。