DNA damage is a constant threat and can be bypassed in a process called translesion synthesis, which is typically carried out by Y-family DNA polymerases. Y-family DNA polymerases are conserved in all domains of life and tend to have specificity for certain types of DNA damage. Escherichia coli DinB and its human ortholog pol κ can bypass specific minor groove deoxyguanine adducts efficiently and are inhibited by major groove adducts, as Y-family DNA polymerases make contacts with the minor groove side of the DNA substrate and lack contacts with the major groove at the nascent base pair. DinB is inhibited by major groove adducts more than pol κ, and they each have active site loops of different lengths, with four additional amino acids in the DinB loop. We previously showed that the R35A active site loop mutation in DinB allows for bypass of the major groove adduct N⁶-furfuryl-dA. These observations led us to investigate the different active site loops by creating loop swap chimeras of DinB with a pol κ loop and vice versa by changing the loop residues in a stepwise fashion. We then determined their activity with undamaged DNA or DNA containing N²-furfuryl-dG or N⁶-furfuryl-dA. The DinB proteins with the pol kappa loop have low activity on all templates but have decreased misincorporation compared to either wild-type protein. The kappa proteins with the DinB loop retain activity on all templates and have decreased misincorporation compared to either wild-type protein. We assessed the thermal stability of the proteins and observed an increase in stability in the presence of all DNA templates and additional increases generally only in the presence of the undamaged and N²-furfuryl-dG adduct and dCTP, which correlates with activity. Overall we find that pol κ is more tolerant to changes in the active site loop than DinB.

中文翻译：

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人Y家族DNA聚合酶κ比其直系同源大肠杆菌DinB更能容忍其活动位点环的变化

DNA损伤是一个持续的威胁，可以在称为“病灶合成”的过程中绕开，该过程通常由Y家族DNA聚合酶进行。Y族DNA聚合酶在生活的所有域中都是保守的，并且倾向于对某些类型的DNA损伤具有特异性。大肠杆菌DinB及其人类直系同源物polκ可以有效绕过特定的小沟脱氧鸟嘌呤加合物，并被大沟加合物抑制，因为Y族DNA聚合酶与DNA底物的小沟侧接触，而与新生时的大沟缺乏接触。碱基对。DinB受到主要沟加合物的抑制作用大于polκ，并且每个都具有不同长度的活性位点环，DinB环中还有四个其他氨基酸。我们先前表明，DinB中的R35A活性位点环突变允许绕开主要凹槽加合物N ⁶-糠基-dA。这些观察结果使我们能够通过创建具有polκ环的DinB的环交换嵌合体来研究不同的活性位点环，反之亦然，方法是通过逐步改变环残基来实现。然后，我们确定了它们在未损坏的DNA或含有N ^2-糠基-dG或N ^6的DNA中的活性。-糠基-dA。具有pol kappa环的DinB蛋白在所有模板上均具有较低的活性，但与任一种野生型蛋白相比，其掺入错误均减少。与任一野生型蛋白相比，具有DinB环的kappa蛋白在所有模板上均保持活性，并减少了错误掺入。我们评估了蛋白质的热稳定性，并观察到在所有DNA模板存在的情况下稳定性的增加，并且通常仅在未破坏的N ^2-糠基dG加合物和dCTP的存在下才增加了活性，这与活性相关。总的来说，我们发现polκ比DinB更能容忍活动位点环中的变化。