Residues located in the primase domain of the bacteriophage T7 primase-helicase are essential for loading the hexameric complex onto DNA.
Residues located in the primase domain of the bacteriophage T7 primase-helicase are essential for loading the hexameric complex onto DNA.
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DOI：
10.1016/j.jbc.2022.101996
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发表时间：
2022-06
影响因子：
4.8
通讯作者：
Richardson, Charles C.
中科院分区：
文献类型：
--
作者：
Hernandez, Alfredo J.;Lee, Seung-Joo;Thompson, Noah J.;Griffith, Jack D.;Richardson, Charles C.
T7引发酶 - 解旋酶在T7 DNA的复制中起着关键作用。通过肽 - 核酸交联的亲和分离和质谱分析，我们确定了引发酶 - 解旋酶和T7 DNA聚合酶中与RNA引物和DNA模板形成接触的蛋白质区域。核酸与引发酶 - 解旋酶的引发酶结构域之间的接触集中在引发酶结构域的RNA聚合酶亚结构域内，位于N - 末端亚结构域和拓扑异构酶 - 引发酶折叠结构之间的裂隙中。我们证明了N - 末端亚结构域中与RNA引物接触的β折叠上的残基对噬菌体生长和体外引发酶活性至关重要。令人惊讶的是，我们发现引发酶结构域中的突变对解旋酶有显著影响。在其他类似DnaG的酶中保守的残基R84A被替换后，T7引发酶 - 解旋酶的引发酶和解旋酶酶活性均丧失。该残基的改变也降低了引发酶 - 解旋酶与单链DNA的结合。然而，质量光度测量表明这些突变并不干扰蛋白质形成有活性六聚体的能力。
The T7 primase-helicase plays a pivotal role in the replication of T7 DNA. Using affinity isolation of peptide–nucleic acid crosslinks and mass spectrometry, we identify protein regions in the primase-helicase and T7 DNA polymerase that form contacts with the RNA primer and DNA template. The contacts between nucleic acids and the primase domain of the primase-helicase are centered in the RNA polymerase subdomain of the primase domain, in a cleft between the N-terminal subdomain and the topoisomerase-primase fold. We demonstrate that residues along a beta sheet in the N-terminal subdomain that contacts the RNA primer are essential for phage growth and primase activity in vitro. Surprisingly, we found mutations in the primase domain that had a dramatic effect on the helicase. Substitution of a residue conserved in other DnaG-like enzymes, R84A, abrogates both primase and helicase enzymatic activities of the T7 primase-helicase. Alterations in this residue also decrease binding of the primase-helicase to ssDNA. However, mass photometry measurements show that these mutations do not interfere with the ability of the protein to form the active hexamer.
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影响因子：
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作者：
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通讯作者：
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DOI：
10.1073/pnas.1604894113
发表时间：
2016-05-24
影响因子：
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