Structures of a DNA Polymerase Inserting Therapeutic Nucleotide Analogues.
Structures of a DNA Polymerase Inserting Therapeutic Nucleotide Analogues.
复制标题
插入治疗性核苷酸类似物的 DNA 聚合酶的结构。
DOI：
10.1021/acs.chemrestox.7b00173
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发表时间：
2017
影响因子：
4.1
通讯作者：
Freudenthal,BretD
中科院分区：
文献类型：
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作者：
Schaich,MatthewA;Smith,MalloryR;Cloud,AshleyS;Holloran,SeanM;Freudenthal,BretD
核苷类似物类癌症治疗剂的成员与规范核苷酸竞争以破坏许多细胞过程，包括核苷酸稳态、DNA和RNA合成以及核苷酸代谢。核苷类似物被三磷酸化并随后插入基因组DNA中，以多种方式促进治疗性核苷的功效。在某些情况下，改变的碱基作为诱变剂，改变DNA序列以促进细胞死亡;在其他情况下，插入改变的核苷酸触发DNA修复途径，其产生致死水平的细胞毒性中间体，如单链和双链DNA断裂。作为许多这些生物学结果的先决条件，修饰的核苷酸必须在核苷酸插入期间容纳在DNA聚合酶活性位点中。目前，介导DNA聚合酶插入修饰的核苷酸的分子接触对于多种治疗化合物仍然是未知的，尽管几十年的临床使用。为了确定修饰的碱基是如何插入双链DNA的，我们使用哺乳动物DNA聚合酶β（pol β）来显示四种与治疗相关的修饰核苷酸的结构构象，6-硫代-2 ′-脱氧鸟苷-5 ′-三磷酸（6-TdGTP），5-氟-2 ′-脱氧尿苷-5 ′-三磷酸（5-FdUTP），5-甲酰基-脱氧胞嘧啶-5 ′-三磷酸（5-FdUTP），（5-FodCTP）和5-甲酰基-脱氧尿苷-5 ′-三磷酸（5-FodUTP）。总之，这些结构揭示了一种模式，其中修饰的核苷酸利用与未修饰的核苷酸类似的沃森-克里克碱基配对相互作用。核苷酸修饰始终位于双链体DNA的大沟中，由pol β中的开放腔容纳。这些结果为合理设计新的治疗性核苷类似物提供了新的信息，并更好地了解聚合酶如何耐受修饰的核苷酸。
Members of the nucleoside analogue class of cancer therapeutics compete with canonical nucleotides to disrupt numerous cellular processes, including nucleotide homeostasis, DNA and RNA synthesis, and nucleotide metabolism. Nucleoside analogues are triphosphorylated and subsequently inserted into genomic DNA, contributing to the efficacy of therapeutic nucleosides in multiple ways. In some cases, the altered base acts as a mutagen, altering the DNA sequence to promote cellular death; in others, insertion of the altered nucleotide triggers DNA repair pathways, which produce lethal levels of cytotoxic intermediates such as single and double stranded DNA breaks. As a prerequisite to many of these biological outcomes, the modified nucleotide must be accommodated in the DNA polymerase active site during nucleotide insertion. Currently, the molecular contacts that mediate DNA polymerase insertion of modified nucleotides remain unknown for multiple therapeutic compounds, despite decades of clinical use. To determine how modified bases are inserted into duplex DNA, we used mammalian DNA polymerase β (pol β) to visualize the structural conformations of four therapeutically relevant modified nucleotides, 6-thio-2′-deoxyguanosine-5′-triphosphate (6-TdGTP), 5-fluoro-2′-deoxyuridine-5′-triphosphate (5-FdUTP), 5-formyl-deoxycytosine-5′-triphosphate (5-FodCTP), and 5-formyl-deoxyuridine-5′-triphosphate (5-FodUTP). Together, the structures reveal a pattern in which the modified nucleotides utilize Watson–Crick base pairing interactions similar to that of unmodified nucleotides. The nucleotide modifications were consistently positioned in the major groove of duplex DNA, accommodated by an open cavity in pol β. These results provide novel information for the rational design of new therapeutic nucleoside analogues and a greater understanding of how modified nucleotides are tolerated by polymerases.