Laboratory of Structural Biology, National Institute of Environmental Health Sciences , Research Triangle Park, North Carolina 27709, United States.
Biochemistry. 2014 Apr 15;53(14):2413-22. doi: 10.1021/bi5001855. Epub 2014 Apr 2.
DNA polymerase (pol) β is a multidomain enzyme with two enzymatic activities that plays a central role in the overlapping base excision repair and single-strand break repair pathways. The high frequency of pol β variants identified in tumor-derived tissues suggests a possible role in the progression of cancer, making the determination of the functional consequences of these variants of interest. Pol β containing a proline substitution for leucine 22 in the lyase domain (LD), identified in gastric tumors, has been reported to exhibit severe impairment of both lyase and polymerase activities. Nuclear magnetic resonance (NMR) spectroscopic evaluations of both pol β and the isolated LD containing the L22P mutation demonstrate destabilization sufficient to result in LD-selective unfolding with minimal structural perturbations to the polymerase domain. Unexpectedly, addition of single-stranded or hairpin DNA resulted in partial refolding of the mutated lyase domain, both in isolation and for the full-length enzyme. Further, formation of an abortive ternary complex using Ca(2+) and a complementary dNTP indicates that the fraction of pol β(L22P) containing the folded LD undergoes conformational activation similar to that of the wild-type enzyme. Kinetic characterization of the polymerase activity of L22P pol β indicates that the L22P mutation compromises DNA binding, but nearly wild-type catalytic rates can be observed at elevated substrate concentrations. The organic osmolyte trimethylamine N-oxide (TMAO) is similarly able to induce folding and kinetic activation of both polymerase and lyase activities of the mutant. Kinetic data indicate synergy between the TMAO cosolvent and substrate binding. NMR data indicate that the effect of the DNA results primarily from interaction with the folded LD(L22P), while the effect of the TMAO results primarily from destabilization of the unfolded LD(L22P). These studies illustrate that substrate-induced catalytic activation of pol β provides an optimal enzyme conformation even in the presence of a strongly destabilizing point mutation. Accordingly, it remains to be determined whether this mutation alters the threshold of cellular repair activity needed for routine genome maintenance or whether the "inactive" variant interferes with DNA repair.
DNA 聚合酶（pol）β 是一种具有两种酶活性的多功能酶，在碱基切除修复和单链断裂修复途径的重叠中发挥核心作用。在肿瘤衍生组织中鉴定出的 pol β 变体的高频率表明其在癌症进展中可能起作用，因此确定这些变体的功能后果很有意义。在胃肿瘤中鉴定出的裂解酶结构域（LD）中脯氨酸取代亮氨酸 22 的 pol β 含有脯氨酸取代亮氨酸 22，已报道其裂解酶和聚合酶活性均严重受损。对 pol β 和含有 L22P 突变的分离 LD 的核磁共振（NMR）光谱评估表明，其足够不稳定，导致 LD 选择性展开，聚合酶结构域的结构扰动最小。出乎意料的是，添加单链或发夹 DNA 会导致突变的裂解酶结构域部分折叠，无论是在分离状态还是在全长酶中。此外，使用 Ca（2+）和互补的 dNTP 形成一个无活性的三元复合物表明，包含折叠 LD 的 pol β（L22P）的分数经历类似于野生型酶的构象激活。L22P pol β 的聚合酶活性的动力学特征表明，L22P 突变会损害 DNA 结合，但在升高的底物浓度下可以观察到几乎野生型的催化速率。有机渗透剂三甲胺 N-氧化物（TMAO）也能够诱导突变体的聚合酶和裂解酶活性的折叠和动力学激活。动力学数据表明 TMAO 共溶剂和底物结合之间存在协同作用。NMR 数据表明，DNA 的影响主要来自与折叠 LD（L22P）的相互作用，而 TMAO 的影响主要来自未折叠 LD（L22P）的失稳。这些研究表明，底物诱导的 pol β 催化激活提供了最佳的酶构象，即使存在强烈的稳定化点突变也是如此。因此，仍有待确定该突变是否改变了维持常规基因组所需的细胞修复活性的阈值，或者“无活性”变体是否干扰 DNA 修复。
