Molecular basis for the initiation of DNA primer synthesis.
Molecular basis for the initiation of DNA primer synthesis.
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DOI：
10.1038/s41586-022-04695-0
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发表时间：
2022-05
期刊：
影响因子：
64.8
通讯作者：
中科院分区：
文献类型：
--
作者：
在 DNA 复制起始期间，寡核苷酸引物由引物酶从头合成，随后由复制聚合酶延伸以完成基因组复制。引物酶聚合酶 (Prim-Pol) 超家族是一个多样化的引物酶群体，其中包括参与适应性免疫的复制引物酶和 CRISPR 相关引物酶聚合酶 (CAPP)。尽管人们对这些酶的活性了解很多，但引物酶用于启动引物合成的精确机制尚未阐明。在这里，我们鉴定了 CAPP 启动引物合成的分子基础，并表明这种机制在复制引物酶中也是保守的。引物起始复合物的晶体结构揭示了在合成第一个磷酸二酯键之前，引入的核苷酸如何定位在活性位点内、邻近金属辅因子并与模板单链 DNA 链配对。此外，具有双链 DNA 的 Prim-Pol 复合物的结构显示了该酶随后如何以持续聚合酶模式延伸引物。本文介绍的结构和机制研究确定了 Prim-Pol 蛋白如何促进引物合成，揭示了催化域内引物合成所需的分子决定因素。这项工作还确定了 Prim-Pol 酶（包括复制引物酶）的催化结构域足以催化引物形成。
During the initiation of DNA replication, oligonucleotide primers are synthesized de novo by primases and are subsequently extended by replicative polymerases to complete genome duplication. The primase-polymerase (Prim-Pol) superfamily is a diverse grouping of primases, which includes replicative primases and CRISPR-associated primase-polymerases (CAPPs) involved in adaptive immunity. Although much is known about the activities of these enzymes, the precise mechanism used by primases to initiate primer synthesis has not been elucidated. Here we identify the molecular bases for the initiation of primer synthesis by CAPP and show that this mechanism is also conserved in replicative primases. The crystal structure of a primer initiation complex reveals how the incoming nucleotides are positioned within the active site, adjacent to metal cofactors and paired to the templating single-stranded DNA strand, before synthesis of the first phosphodiester bond. Furthermore, the structure of a Prim-Pol complex with double-stranded DNA shows how the enzyme subsequently extends primers in a processive polymerase mode. The structural and mechanistic studies presented here establish how Prim-Pol proteins instigate primer synthesis, revealing the requisite molecular determinants for primer synthesis within the catalytic domain. This work also establishes that the catalytic domain of Prim-Pol enzymes, including replicative primases, is sufficient to catalyse primer formation.
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