Although translesion synthesis (TLS) polymerases play key roles in replicating DNA that contains nucleobase addition products (adducts), there are many unknowns about their function. The present work gains indispensable structural insights from molecular dynamics simulations on the replication of O6-benzyl-guanine (Bz-dG) prior to bond formation during dCTP insertion opposite the adduct by Dpo4. When combined with previous X-ray crystal structures of the Bz-dG extension complex, molecular details are now available for each stage during a single TLS replication cycle for this carcinogenic lesion. Our calculations illustrate that Bz-dG preferentially adopts an intercalated bulky moiety orientation in the Dpo4 preinsertion complex, which stabilizes the complex through Bz-dG interactions with the previously replicated 3′-base pair and positions the carcinogenic group in the dNTP binding site. Nevertheless, the maintained inherent flexibility of Bz-dG due to a stark lack of interactions with the polymerase or template DNA allows the bulky moiety to adopt a major groove position opposite an incoming dCTP in an orientation that is conducive for the experimentally observed nonmutagenic bypass. Comparison of Bz-dG and canonical dG replication clarifies that the experimentally observed decrease in dCTP binding affinity and replication efficiency upon adduct formation is likely caused by a combination of factors, including the required template nucleotide conformational change and destabilized template–dCTP hydrogen bonding. Although additional aspects of the replication process, such as the impact of the adduct on the nucleotidyl-transfer reaction, may also be important for fully rationalizing experimental replication data and must be considered in future work, the present contribution emphasizes the importance of considering the effect of DNA damage on the early stages of the TLS process.

中文翻译：

---

旁路聚合酶活性位点中苄基-鸟嘌呤加合物的构象柔性允许复制：分子动力学模拟的见解

尽管跨病变合成（TLS）聚合酶在复制包含核碱基加成产物（加合物）的DNA中起着关键作用，但是关于其功能仍有许多未知数。当前的工作从分子动力学模拟获得了必不可少的结构见解，这些分子动力学模拟是在dCTP插入过程中与Dpo4加合物相对的键形成之前，O6-苄基鸟嘌呤（Bz-dG）的复制。当与Bz-dG延伸复合物的先前X射线晶体结构结合时，该致癌性病变的单个TLS复制周期中的每个阶段的分子详细信息现在即可使用。我们的计算表明，Bz-dG在Dpo4预插入复合物中优先采用插入的大块部分取向，它通过与先前复制的3'-碱基对的Bz-dG相互作用稳定复合物，并将致癌基团定位在dNTP结合位点。然而，由于与聚合酶或模板DNA的相互作用明显缺乏相互作用而保持的Bz-dG固有的柔韧性允许大体积部分在与引入的dCTP相对的主要凹槽位置采用有利于实验观察到的非诱变旁路的方向。Bz-dG和经典dG复制的比较阐明，实验观察到的加合物形成后dCTP结合亲和力和复制效率的降低可能是由多种因素共同导致的，包括所需的模板核苷酸构象变化和不稳定的模板-dCTP氢键。尽管复制过程还有其他方面，