BACKGROUND Formamidopyrimidine-DNA glycosylase (Fpg) removes abundant pre-mutagenic 8-oxoguanine (oxoG) bases from DNA through nucleophilic attack of its N-terminal proline at C1' of the damaged nucleotide. Since oxoG efficiently pairs with both C and A, Fpg must excise oxoG from pairs with C but not with A, otherwise a mutation occurs. The crystal structures of several Fpg-DNA complexes have been solved, yet no structure with A opposite the lesion is available. RESULTS Here we use molecular dynamic simulation to model interactions in the pre-catalytic complex of Lactococcus lactis Fpg with DNA containing oxoG opposite C or A, the latter in either syn or anti conformation. The catalytic dyad, Pro1-Glu2, was modeled in all four possible protonation states. Only one transition was observed in the experimental reaction rate pH dependence plots, and Glu2 kept the same set of interactions regardless of its protonation state, suggesting that it does not limit the reaction rate. The adenine base opposite oxoG was highly distorting for the adjacent nucleotides: in the more stable syn models it formed non-canonical bonds with out-of-register nucleotides in both the damaged and the complementary strand, whereas in the anti models the adenine either formed non-canonical bonds or was expelled into the major groove. The side chains of Arg109 and Phe111 that Fpg inserts into DNA to maintain its kinked conformation tended to withdraw from their positions if A was opposite to the lesion. The region showing the largest differences in the dynamics between oxoG:C and oxoG:A substrates was unexpectedly remote from the active site, located near the linker joining the two domains of Fpg. This region was also highly conserved among 124 analyzed Fpg sequences. Three sites trapping water molecules through multiple bonds were identified on the protein-DNA interface, apparently helping to maintain enzyme-induced DNA distortion and participating in oxoG recognition. CONCLUSION Overall, the discrimination against A opposite to the lesion seems to be due to incorrect DNA distortion around the lesion-containing base pair and, possibly, to gross movement of protein domains connected by the linker.

中文翻译：

---

反向动力学偏好和甲酰胺嘧啶-DNA糖基化酶活性位点相互作用的分子动力学模拟。

背景技术甲酰嘧啶-DNA糖基化酶（Fpg）通过其N末端脯氨酸在受损核苷酸的C1'处的亲核攻击而从DNA中去除了丰富的诱变前8-氧鸟嘌呤（oxoG）碱基。由于oxoG有效地与C和A配对，因此Fpg必须从与C但与A配对的中切除oxoG，否则会发生突变。已经解决了几种Fpg-DNA复合物的晶体结构，但尚无与病变相反的A结构。结果在这里，我们使用分子动力学模拟来模拟乳酸乳球菌Fpg的预催化复合物与含有与C或A相对的oxoG的DNA的相互作用，后者为同构或反构象。在所有四个可能的质子化状态下对催化二元组Pro1-Glu2进行建模。在实验反应速率pH依赖性图中仅观察到一个转变，Glu2和Glu2保持相同的相互作用集，而不管其质子化状态如何，这表明它不限制反应速率。与oxoG相对的腺嘌呤碱基对相邻核苷酸高度扭曲：在更稳定的syn模型中，它在受损链和互补链中均形成具有非套准核苷酸的非规范键，而在反模型中，腺嘌呤要么形成非规范键或被驱逐到主要凹槽中。Fpg插入DNA中以维持其扭结构象的Arg109和Phe111的侧链，如果A与病变相反，则倾向于从其位置退出。oxoG：C和oxoG：A底物之间动力学差异最大的区域出乎意料地远离活性位点，位于连接Fpg两个结构域的接头附近。该区域在124个分析的Fpg序列中也高度保守。在蛋白质-DNA界面上确定了通过多个键捕获水分子的三个位点，显然有助于维持酶诱导的DNA畸变并参与oxoG识别。结论总的来说，对与病变相反的A的歧视似乎是由于含病变的碱基对周围的DNA扭曲不正确，以及可能是由连接子连接的蛋白质结构域的总体运动所致。