Members of the conserved Argonaute (Ago) protein family provide defence against invading nucleic acids in eukaryotes in the process of RNA interference. Many prokaryotes also contain Ago proteins that are predicted to be active nucleases; however, their functional activities in host cells remain poorly understood. Here, we characterize the in vitro and in vivo properties of the SeAgo protein from the mesophilic cyanobacterium Synechococcus elongatus. We show that SeAgo is a DNA-guided nuclease preferentially acting on single-stranded DNA targets, with non-specific guide-independent activity observed for double-stranded substrates. The SeAgo gene is steadily expressed in S. elongatus; however, its deletion or overexpression does not change the kinetics of cell growth. When purified from its host cells or from heterologous E. coli, SeAgo is loaded with small guide DNAs whose formation depends on the endonuclease activity of the argonaute protein. SeAgo co-purifies with SSB proteins suggesting that they may also be involved in DNA processing. The SeAgo-associated small DNAs are derived from diverse genomic locations, with certain enrichment for the proposed sites of chromosomal replication initiation and termination, but show no preference for an endogenous plasmid. Therefore, promiscuous genome sampling by SeAgo does not have great effects on cell physiology and plasmid maintenance.

中文翻译：

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通过Ago核酸酶从蓝细菌Synchococcus elongatus进行全基因组DNA采样。

保守的Argonaute（Ago）蛋白家族的成员在RNA干扰过程中提供了对真核生物中入侵核酸的防御作用。许多原核生物还含有Ago蛋白，该蛋白被认为是活性核酸酶。然而，它们在宿主细胞中的功能活性仍然知之甚少。在这里，我们表征了来自嗜温性蓝藻Synchococcus elongatus的SeAgo蛋白的体外和体内特性。我们显示，SeAgo是一种DNA指导的核酸酶，优先作用于单链DNA靶标，对双链底物具有非特异性的指导独立活性。SeAgo基因在伸长链球菌中稳定表达；但是，其缺失或过表达不会改变细胞生长的动力学。从其宿主细胞或异源大肠杆菌中纯化后，SeAgo上装有小的指导DNA，其形成取决于精氨酸蛋白的核酸内切酶活性。SeAgo与SSB蛋白共纯化，表明它们也可能参与DNA加工。与SeAgo相关的小DNA来源于不同的基因组位置，对于拟议的染色体复制起始和终止位点有一定的富集，但对内源质粒没有任何偏好。因此，通过SeAgo进行混杂基因组采样不会对细胞生理和质粒维持产生重大影响。具有建议的染色体复制起始和终止位点的某些富集，但对内源质粒没有偏好。因此，通过SeAgo进行混杂基因组采样不会对细胞生理和质粒维持产生重大影响。具有建议的染色体复制起始和终止位点的某些富集，但对内源质粒没有偏好。因此，通过SeAgo进行混杂基因组采样不会对细胞生理和质粒维持产生重大影响。