The function of many DNA processing enzymes involves sliding along the double helix or individual DNA strands. Stable secondary structures in the form of G-quadruplexes are difficult for such enzymes to bypass. We used a polymerase stop assay to determine which structural features of the human telomeric and the BCL2 promoter G-quadruplexes can stall progression of the Klenow fragment. Primer extension profiles revealed that G-quartets are effective roadblocks for the Klenow fragment, while auxiliary base pairs can be easily bypassed. Furthermore, we utilized 8-oxoguanine to simulate oxidative damage in G-rich regions and determine the effects on enzyme bypass. In rare cases, oxidative lesions reduce the level of G-quadruplex bypass. In general, however, oxidative lesions reduce G-quadruplex stability and facilitate bypassing of such G-rich regions, especially if the lesion persists in unfolding intermediates. Our findings using Klenow fragment can be extrapolated to other G-quadruplex forming sequences and enzymes that utilise a clamp-like structure to slide along DNA and are involved in processes such as gene expression regulation and telomere maintenance.

中文翻译：

---

包含氧化损伤的 G-四链体结构的酶旁路


许多 DNA 加工酶的功能包括沿双螺旋或单个 DNA 链滑动。这种酶很难绕过 G-四链体形式的稳定二级结构。我们使用聚合酶终止测定来确定人端粒和 BCL2 启动子 G-四链体的哪些结构特征可以阻止 Klenow 片段的进展。引物延伸曲线显示，G 四重奏是 Klenow 片段的有效障碍，而辅助碱基对可以很容易地绕过。此外，我们利用 8-氧代鸟嘌呤来模拟富含 G 区域的氧化损伤并确定对酶旁路的影响。在极少数情况下，氧化性病变会降低 G 四链体旁路的水平。然而，一般来说，氧化性病变会降低 G 四链体的稳定性，并有助于绕过这些富含 G 的区域，特别是如果病变持续存在于展开的中间体中。我们使用 Klenow 片段的发现可以外推到其他 G 四链体形成序列和酶，这些序列和酶利用钳状结构沿 DNA 滑动，并参与基因表达调节和端粒维持等过程。