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首页> 外文期刊>Journal of Molecular Biology >Insights into eukaryotic primer synthesis from structures of the p48 subunit of human DNA primase
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Insights into eukaryotic primer synthesis from structures of the p48 subunit of human DNA primase

机译：从人类DNA primase的p48亚基结构了解真核引物合成

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摘要

DNA replication in all organisms requires polymerases to synthesize copies of the genome. DNA polymerases are unable to function on a bare template and require a primer. Primases are crucial RNA polymerases that perform the initial de novo synthesis, generating the first 8-10 nucleotides of the primer. Although structures of archaeal and bacterial primases have provided insights into general priming mechanisms, these proteins are not well conserved with heterodimeric (p48/p58) primases in eukaryotes. Here, we present X-ray crystal structures of the catalytic engine of a eukaryotic primase, which is contained in the p48 subunit. The structures of p48 reveal that eukaryotic primases maintain the conserved catalytic prim fold domain, but with a unique subdomain not found in the archaeal and bacterial primases. Calorimetry experiments reveal that Mn2 + but not Mg2 + significantly enhances the binding of nucleotide to primase, which correlates with higher catalytic efficiency in vitro. The structure of p48 with bound UTP and Mn2 + provides insights into the mechanism of nucleotide synthesis by primase. Substitution of conserved residues involved in either metal or nucleotide binding alter nucleotide binding affinities, and yeast strains containing the corresponding Pri1p substitutions are not viable. Our results reveal that two residues (S160 and H166) in direct contact with the nucleotide were previously unrecognized as critical to the human primase active site. Comparing p48 structures to those of similar polymerases in different states of action suggests changes that would be required to attain a catalytically competent conformation capable of initiating dinucleotide synthesis.
机译：在所有生物中的DNA复制都需要聚合酶来合成基因组的副本。 DNA聚合酶无法在裸模板上起作用，需要引物。引发酶是至关重要的RNA聚合酶，可进行从头合成，产生引物的前8-10个核苷酸。尽管古细菌和细菌引发酶的结构已为一般引发机制提供了见识，但这些蛋白与真​​核生物中的异二聚体（p48 / p58）引发酶并不十分保守。在这里，我们介绍了真核生物引发酶的催化引擎的X射线晶体结构，该结构包含在p48亚基中。 p48的结构表明，真核启动酶保留了保守的催化启动子折叠结构域，但具有古细菌和细菌启动酶中未发现的独特子结构域。量热实验表明，Mn2 +而不是Mg2 +显着增强核苷酸与primase的结合，这与更高的体外催化效率相关。具有结合的UTP和Mn2 +的p48的结构提供了对由primase合成核苷酸的机制的认识。取代涉及金属或核苷酸结合的保守残基会改变核苷酸结合亲和力，并且含有相应Pri1p取代的酵母菌株不可行。我们的结果表明，与核苷酸直接接触的两个残基（S160和H166）以前未被认为对人类primase活性位点至关重要。将p48结构与处于不同作用状态的类似聚合酶的p48结构进行比较，表明需要改变才能获得能够启动二核苷酸合成的催化有效构象。

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