Department of Chemistry, University of Pittsburgh, Pittsburgh, PA 15260, USA.
Biochemistry. 2011 Jun 14;50(23):5379-90. doi: 10.1021/bi200421g. Epub 2011 May 17.
DNA replication polymerases have the inherent ability to faithfully and rapidly copy a DNA template according to precise Watson-Crick base pairing. The primary B-family DNA replication polymerase (Dpo1) in the hyperthermophilic archaeon, Sulfolobus solfataricus, is shown here to possess a remarkable DNA stabilizing ability for maintaining weak base pairing interactions to facilitate primer extension. This thermal stabilization by Dpo1 allowed for template-directed synthesis at temperatures more than 30 °C above the melting temperature of naked DNA. Surprisingly, Dpo1 also displays a competing terminal deoxynucleotide transferase (TdT) activity unlike any other B-family DNA polymerase. Dpo1 is shown to elongate single-stranded DNA in template-dependent and template-independent manners. Experiments with different homopolymeric templates indicate that initial deoxyribonucleotide incorporation is complementary to the template. Rate-limiting steps that include looping back and annealing to the template allow for a unique template-dependent terminal transferase activity. The multiple activities of this unique B-family DNA polymerase make this enzyme an essential component for DNA replication and DNA repair for the maintenance of the archaeal genome at high temperatures.
DNA 复制聚合酶具有根据精确的沃森-克里克碱基配对忠实而快速复制 DNA 模板的固有能力。嗜热古菌 Sulfolobus solfataricus 中的主要 B 族 DNA 复制聚合酶（Dpo1）具有显著的 DNA 稳定能力，可维持弱碱基配对相互作用，从而促进引物延伸。Dpo1 的这种热稳定性允许在比裸露 DNA 熔点高 30°C 以上的温度下进行模板指导的合成。令人惊讶的是，Dpo1 还表现出不同于任何其他 B 族 DNA 聚合酶的竞争末端脱氧核苷酸转移酶（TdT）活性。研究表明，Dpo1 以模板依赖性和模板非依赖性方式延伸单链 DNA。使用不同的均聚物模板的实验表明，最初的脱氧核苷酸掺入与模板互补。包括回环和与模板退火的限速步骤允许独特的模板依赖性末端转移酶活性。这种独特的 B 族 DNA 聚合酶的多种活性使其成为在高温下维持古菌基因组的 DNA 复制和 DNA 修复的必需组成部分。
