Rudolf Virchow Center for Experimental Biomedicine, Würzburg, Germany.
DNA Repair (Amst). 2012 Mar 1;11(3):286-93. doi: 10.1016/j.dnarep.2011.12.002. Epub 2012 Jan 9.
Bax1 has recently been identified as a novel binding partner for the archaeal helicase XPB. We previously characterized Bax1 from Thermoplasma acidophilum as a Mg²⁺-dependent structure-specific endonuclease. Here we directly compare the endonuclease activity of Bax1 alone or in combination with XPB. Using several biochemical and biophysical approaches, we demonstrate regulation of Bax1 endonuclease activity by XPB. Interestingly, incision assays with Bax1 and XPB/Bax1 clearly demonstrate that Bax1 produces different incision patterns depending on the presence or absence of XPB. Using atomic force microscopy (AFM), we directly visualize and compare binding of Bax1 and XPB/Bax1 to different DNA substrates. Our AFM data support enhanced DNA binding affinity of Bax1 in the presence of XPB. Taken together, the DNA incision and binding results suggest that XPB is able to load and position Bax1 on the scissile DNA substrate, thus increasing the DNA substrate range of Bax1.
Bax1 最近被鉴定为古菌解旋酶 XPB 的一个新结合伴侣。我们之前从嗜酸热原体中鉴定出 Bax1 是一种依赖于 Mg²⁺的结构特异性内切核酸酶。在这里，我们直接比较了 Bax1 单独或与 XPB 组合的内切核酸酶活性。使用几种生化和生物物理方法，我们证明了 XPB 对 Bax1 内切核酸酶活性的调节。有趣的是，使用 Bax1 和 XPB/Bax1 的切口分析清楚地表明，Bax1 根据 XPB 的存在与否产生不同的切口模式。使用原子力显微镜 (AFM)，我们直接可视化和比较了 Bax1 和 XPB/Bax1 与不同 DNA 底物的结合。我们的 AFM 数据支持在 XPB 存在下 Bax1 具有增强的 DNA 结合亲和力。综上所述，DNA 切口和结合结果表明，XPB 能够将 Bax1 加载并定位到切口 DNA 底物上，从而增加了 Bax1 的 DNA 底物范围。
