Sampoli Benítez Benedetta A, Arora Karunesh, Balistreri Lisa, Schlick Tamar
Department of Natural Sciences and Mathematics, Marymount Manhattan College, 221 East 71st Street, New York, NY 10021, USA.
J Mol Biol. 2008 Dec 31;384(5):1086-97. doi: 10.1016/j.jmb.2008.10.025. Epub 2008 Oct 17.
DNA polymerase X (pol X) from the African swine fever virus is a 174-amino-acid repair polymerase that likely participates in a viral base excision repair mechanism, characterized by low fidelity. Surprisingly, pol X's insertion rate of the GG mispair is comparable to that of the four Watson-Crick base pairs. This behavior is in contrast with another X-family polymerase, DNA polymerase beta (pol beta), which inserts GG mismatches poorly, and has higher DNA repair fidelity. Using molecular dynamics simulations, we previously provided support for an induced-fit mechanism for pol X in the presence of the correct incoming nucleotide. Here, we perform molecular dynamics simulations of pol X/DNA complexes with different incoming incorrect nucleotides in various orientations [CC, AG, and GG (anti) and AG and GG (syn)] and compare the results to available kinetic data and prior modeling. Intriguingly, the simulations reveal that the GG mispair with the incoming nucleotide in the syn configuration undergoes large-scale conformational changes similar to that observed in the presence of correct base pair (GC). The base pairing in the GG mispair is achieved via Hoogsteen hydrogen bonding with an overall geometry that is well poised for catalysis. Simulations for other mismatched base pairs show that an intermediate closed state is achieved for the AG and GG mispair with the incoming dGTP in anti conformation, while the protein remains near the open conformation for the CC and the AG syn mismatches. In addition, catalytic site geometry and base pairing at the nascent template-incoming nucleotide interaction reveal distortions and misalignments that range from moderate for AG anti to worst for the CC complex. These results agree well with kinetic data for pol X and provide a structural/dynamic basis to explain, at atomic level, the fidelity of this polymerase compared with other members of the X family. In particular, the more open and pliant active site of pol X, compared to pol beta, allows pol X to accommodate bulkier mismatches such as guanine opposite guanine, while the more structured and organized pol beta active site imposes higher discrimination, which results in higher fidelity. The possibility of syn conformers resonates with other low-fidelity enzymes such as Dpo4 (from the Y family), which readily accommodate oxidative lesions.
非洲猪瘟病毒的DNA聚合酶X（pol X）是一种由174个氨基酸组成的修复聚合酶，可能参与病毒碱基切除修复机制，其特点是保真度较低。令人惊讶的是，pol X对GG错配的插入率与四种沃森-克里克碱基对的插入率相当。这种行为与另一种X家族聚合酶DNA聚合酶β（pol β）形成对比，后者对GG错配的插入能力较差，且具有更高的DNA修复保真度。我们之前通过分子动力学模拟，为pol X在存在正确进入核苷酸时的诱导契合机制提供了支持。在此，我们对pol X/DNA复合物与不同进入的错误核苷酸在各种取向[CC、AG和GG（反式）以及AG和GG（顺式）]下进行分子动力学模拟，并将结果与现有的动力学数据和先前的建模进行比较。有趣的是，模拟结果表明，与进入核苷酸处于顺式构型的GG错配会发生大规模构象变化，类似于在存在正确碱基对（GC）时观察到的情况。GG错配中的碱基配对是通过Hoogsteen氢键实现的，其整体几何结构有利于催化作用。对其他错配碱基对的模拟表明，对于与进入的处于反式构象的dGTP形成的AG和GG错配，会达到一个中间封闭状态，而对于CC和AG顺式错配，蛋白质则保持在接近开放的构象。此外，新生模板与进入核苷酸相互作用处的催化位点几何结构和碱基配对显示出扭曲和错位，从AG反式的中度到CC复合物的最严重程度不等。这些结果与pol X的动力学数据非常吻合，并提供了一个结构/动力学基础，以便在原子水平上解释这种聚合酶与X家族其他成员相比的保真度。特别是，与pol β相比，pol X的活性位点更开放、更柔韧，这使得pol X能够容纳更大的错配，如鸟嘌呤与鸟嘌呤相对，而结构更规整、组织更有序的pol β活性位点具有更高的辨别能力，从而导致更高的保真度。顺式构象异构体的可能性与其他低保真度酶（如来自Y家族的Dpo4）相呼应，后者能够轻易容纳氧化损伤。
