The ability of mutations to facilitate adaptation is central to evolution. To understand how mutations can lead to functional adaptation in a complex molecular machine, we created a defective version of the T4 clamp-loader complex, which is essential for DNA replication. This variant, which is ∼5000-fold less active than the wildtype, was made by replacing the catalytic domains with those from another phage. A directed-evolution experiment revealed that multiple substitutions to a single negatively-charged residue in the chimeric clamp loader – Asp 86 – restore fitness to within ∼20-fold of wildtype. These mutations remove an adventitious electrostatic repulsive interaction between Asp 86 and the sliding clamp. Deep mutagenesis shows that the reduced fitness of the chimeric clamp loader is compensated for by lysine and arginine substitutions of several DNA-proximal residues in the clamp loader or the sliding clamp. Thus, the fitness decrease of the chimeric clamp loader is caused by a reduction in affinity between the clamp loader and the clamp. Our results demonstrate that there is a latent capacity for increasing affinity of the clamp loader for DNA and the sliding clamp, such that even single point mutations can readily compensate for the loss of function due to suboptimal interactions elsewhere.

中文翻译：

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DNA 聚合酶夹钳装载机 ATP 酶复合物的适应能力

突变促进适应的能力是进化的核心。为了了解突变如何导致复杂分子机器的功能适应，我们创建了 T4 夹钳装载复合物的有缺陷版本，该复合物对于 DNA 复制至关重要。这种变体的活性比野生型低约 5000 倍，是通过用另一种噬菌体的催化结构域替换催化结构域而制成的。一项定向进化实验表明，对嵌合夹具装载机（Asp 86）中单个带负电荷的残基进行多次替换，可将适应性恢复到野生型的约 20 倍之内。这些突变消除了 Asp 86 和滑动夹之间的偶然静电排斥相互作用。深度诱变表明，嵌合钳装载机适应性的降低可以通过钳装载机或滑动钳中几个近端DNA残基的赖氨酸和精氨酸取代来补偿。因此，嵌合夹具加载器的适应性下降是由夹具加载器和夹具之间的亲和力降低引起的。我们的结果表明，存在增加夹具加载器对 DNA 和滑动夹具的亲和力的潜在能力，因此即使是单点突变也可以很容易地补偿由于其他地方的次优相互作用而导致的功能丧失。