Functional and Phylogenetic Diversity of Cas10 Proteins.
Functional and Phylogenetic Diversity of Cas10 Proteins.
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DOI：
10.1089/crispr.2022.0085
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发表时间：
2023-03
期刊：
影响因子：
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通讯作者：
T. Wiegand;R. Wilkinson;Andrew Santiago-Frangos;Mackenzie M. Lynes;R. Hatzenpichler;B. Wiedenheft
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文献类型：
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作者：
T. Wiegand;R. Wilkinson;Andrew Santiago-Frangos;Mackenzie M. Lynes;R. Hatzenpichler;B. Wiedenheft
Cas10蛋白是III型CRISPR RNA(CrRNA)引导的监视复合体的大亚基，其中许多具有核酸酶和环化酶活性。在这里，我们使用计算和系统发育方法从基因组和元基因组数据库中识别和分析2014个Cas10序列。Cas10蛋白分为五个不同的分支，反映了之前建立的CRISPR-CAS亚型。大多数Cas10蛋白(85.0%)具有保守的聚合酶活性位点基序，而HD-核酸酶结构域的保守性较差(36.0%)。我们发现Cas10变异体分裂在多个基因上，或者在基因上与由环核苷酸(即NucC)或毒素-抗毒素系统的组成部分(即AbiEii)激活的核酸酶融合。为了阐明Cas10蛋白的功能多样性，我们从三个不同的系统发育分支中克隆、表达和纯化了五个代表蛋白。分离到的Cas10都不是功能循环酶，用聚合酶结构域活性位点突变体进行的活性分析表明，以前报道的Cas10 DNA聚合酶活性可能是污染的结果。总的来说，这项工作有助于阐明III型CRISPR系统中Cas10蛋白的系统发育和功能多样性。
Cas10 proteins are large subunits of type III CRISPR RNA (crRNA)-guided surveillance complexes, many of which have nuclease and cyclase activities. Here, we use computational and phylogenetic methods to identify and analyze 2014 Cas10 sequences from genomic and metagenomic databases. Cas10 proteins cluster into five distinct clades that mirror previously established CRISPR-Cas subtypes. Most Cas10 proteins (85.0%) have conserved polymerase active-site motifs, while HD-nuclease domains are less well conserved (36.0%). We identify Cas10 variants that are split over multiple genes or genetically fused to nucleases activated by cyclic nucleotides (i.e., NucC) or components of toxin-antitoxin systems (i.e., AbiEii). To clarify the functional diversification of Cas10 proteins, we cloned, expressed, and purified five representatives from three phylogenetically distinct clades. None of the Cas10s are functional cyclases in isolation, and activity assays performed with polymerase domain active site mutants indicate that previously reported Cas10 DNA-polymerase activity may be a result of contamination. Collectively, this work helps clarify the phylogenetic and functional diversity of Cas10 proteins in type III CRISPR systems.