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Argonaute of the archaeon Pyrococcus furiosus is a DNA-guided nuclease that targets cognate DNA.

Swarts DC, Hegge JW, Hinojo I, Shiimori M, Ellis MA, Dumrongkulraksa J, Terns RM, Terns MP, van der Oost J - Nucleic Acids Res. (2015)

Bottom Line: PfAgo utilizes small 5'-phosphorylated DNA guides to cleave both single stranded and double stranded DNA targets, and does not utilize RNA as guide or target.Thus, with respect to function and specificity, the archaeal PfAgo resembles bacterial Argonautes much more than eukaryotic Argonautes.These findings demonstrate that the role of Argonautes is conserved through the bacterial and archaeal domains of life and suggests that eukaryotic Argonautes are derived from DNA-guided DNA-interfering host defense systems.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Microbiology, Department of Agrotechnology and Food Sciences, Wageningen University, 6703 HB Wageningen, The Netherlands.

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Plasmid cleavage by PfAgo. (A) pWUR790 expression plasmid. (B) PfAgo expressed at 20°C and purified in absence of Mn2+ cleaves expression plasmid pWUR790. Agarose gels with plasmid targets incubated without protein (lane 1), with PfAgoDM (lane 2) and with PfAgo (lane 3). M1: 1-kb DNA ladder (New England Biolabs). M2: pWUR790 marker with open circular (OC), linearized (LIN) and supercoiled (SC) pWUR790. (C) pWUR704 target plasmid, target site indicated in gray. (D) Target region (gray) and FW and RV siDNA guides (black). Predicted cleavage sites are indicated with a black triangle. (E) Agarose gels with plasmid targets incubated with PfAgoDM (lane 1), with guide free PfAgo (lane 2) and with PfAgo loaded with FW siDNA, RV siDNA, or both (lane 3–5) in reaction buffer with 250 mM NaCl (left panel) or 500 mM NaCl (right panel). M1: 1 kb GeneRuler marker (Thermo Scientific). M2: pWUR704 marker with open circular (OC), linearized (LIN) and supercoiled (SC) pWUR704.
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Figure 5: Plasmid cleavage by PfAgo. (A) pWUR790 expression plasmid. (B) PfAgo expressed at 20°C and purified in absence of Mn2+ cleaves expression plasmid pWUR790. Agarose gels with plasmid targets incubated without protein (lane 1), with PfAgoDM (lane 2) and with PfAgo (lane 3). M1: 1-kb DNA ladder (New England Biolabs). M2: pWUR790 marker with open circular (OC), linearized (LIN) and supercoiled (SC) pWUR790. (C) pWUR704 target plasmid, target site indicated in gray. (D) Target region (gray) and FW and RV siDNA guides (black). Predicted cleavage sites are indicated with a black triangle. (E) Agarose gels with plasmid targets incubated with PfAgoDM (lane 1), with guide free PfAgo (lane 2) and with PfAgo loaded with FW siDNA, RV siDNA, or both (lane 3–5) in reaction buffer with 250 mM NaCl (left panel) or 500 mM NaCl (right panel). M1: 1 kb GeneRuler marker (Thermo Scientific). M2: pWUR704 marker with open circular (OC), linearized (LIN) and supercoiled (SC) pWUR704.

Mentions: To test whether PfAgo cleaves plasmid DNA, PfAgo was incubated with its expression plasmid pWUR790 (Figure 5A). As incubation of this plasmid at 95°C in the presence of Mn2+ results in degradation of the plasmid (even in the absence of PfAgo), we incubated the reaction mixtures at 75°C for 16 h. Still, the majority of the plasmid DNA is turned to the open circular confirmation under these conditions, even in absence of PfAgo (Figure 5B). Strikingly, pWUR790 is linearized when incubated with PfAgo in absence of guides (Figure 5B). We observed this suspected guide-free PfAgo-mediated cleavage of pWUR790 in buffer with 250 mM NaCl, but not in buffer with 500 mM NaCl (Figure 5B). These findings suggest either that PfAgo cleaves plasmids independently of co-purified DNAs or that DNA guides are utilized but the concentration of such co-purified DNA is below the detection limit of the assay. It has previously been demonstrated that in vivo, TtAgo acquires guides that allow for targeting its expression vector (11). To rule out that this PfAgo activity was guided by co-purified RNA or by co-purified DNA guides that we were unable to detect, we used pWUR704 as target plasmid (Figure 5C). pWUR704 has minimal sequence similarity to the PfAgo expression vector (pWUR790; no sequences longer than 13 consecutive identical base pairs between the two plasmids). After incubation without PfAgo, the plasmid is present both in open circular and supercoiled configuration (Figure 5E, lane 1). When PfAgo is added, supercoiled pWUR704 is linearized even in absence of guides (Figure 5E, lane 2). Like pWUR790 cleavage, this activity is more pronounced at 250 mM NaCl compared to 500 mM NaCl. In contrast, TtAgo, which co-purified with detectable levels of siDNA, was unable to cleave pWUR704, unless synthetic siDNAs targeting pWUR790 were added (3). Low levels of guide-free PfAgo-mediated nicking of pWUR704 takes place within 1 h incubation at 75°C (Supplementary Figure S4). As PfAgo does not mediate RNA-guided activity within the same time span (Figure 4), it is unlikely that the activity on plasmid DNA is mediated by RNA that co-purified with PfAgo. Instead, these findings suggest that PfAgo has the potential to cleave the plasmid independently of guides.


Argonaute of the archaeon Pyrococcus furiosus is a DNA-guided nuclease that targets cognate DNA.

Swarts DC, Hegge JW, Hinojo I, Shiimori M, Ellis MA, Dumrongkulraksa J, Terns RM, Terns MP, van der Oost J - Nucleic Acids Res. (2015)

Plasmid cleavage by PfAgo. (A) pWUR790 expression plasmid. (B) PfAgo expressed at 20°C and purified in absence of Mn2+ cleaves expression plasmid pWUR790. Agarose gels with plasmid targets incubated without protein (lane 1), with PfAgoDM (lane 2) and with PfAgo (lane 3). M1: 1-kb DNA ladder (New England Biolabs). M2: pWUR790 marker with open circular (OC), linearized (LIN) and supercoiled (SC) pWUR790. (C) pWUR704 target plasmid, target site indicated in gray. (D) Target region (gray) and FW and RV siDNA guides (black). Predicted cleavage sites are indicated with a black triangle. (E) Agarose gels with plasmid targets incubated with PfAgoDM (lane 1), with guide free PfAgo (lane 2) and with PfAgo loaded with FW siDNA, RV siDNA, or both (lane 3–5) in reaction buffer with 250 mM NaCl (left panel) or 500 mM NaCl (right panel). M1: 1 kb GeneRuler marker (Thermo Scientific). M2: pWUR704 marker with open circular (OC), linearized (LIN) and supercoiled (SC) pWUR704.
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Figure 5: Plasmid cleavage by PfAgo. (A) pWUR790 expression plasmid. (B) PfAgo expressed at 20°C and purified in absence of Mn2+ cleaves expression plasmid pWUR790. Agarose gels with plasmid targets incubated without protein (lane 1), with PfAgoDM (lane 2) and with PfAgo (lane 3). M1: 1-kb DNA ladder (New England Biolabs). M2: pWUR790 marker with open circular (OC), linearized (LIN) and supercoiled (SC) pWUR790. (C) pWUR704 target plasmid, target site indicated in gray. (D) Target region (gray) and FW and RV siDNA guides (black). Predicted cleavage sites are indicated with a black triangle. (E) Agarose gels with plasmid targets incubated with PfAgoDM (lane 1), with guide free PfAgo (lane 2) and with PfAgo loaded with FW siDNA, RV siDNA, or both (lane 3–5) in reaction buffer with 250 mM NaCl (left panel) or 500 mM NaCl (right panel). M1: 1 kb GeneRuler marker (Thermo Scientific). M2: pWUR704 marker with open circular (OC), linearized (LIN) and supercoiled (SC) pWUR704.
Mentions: To test whether PfAgo cleaves plasmid DNA, PfAgo was incubated with its expression plasmid pWUR790 (Figure 5A). As incubation of this plasmid at 95°C in the presence of Mn2+ results in degradation of the plasmid (even in the absence of PfAgo), we incubated the reaction mixtures at 75°C for 16 h. Still, the majority of the plasmid DNA is turned to the open circular confirmation under these conditions, even in absence of PfAgo (Figure 5B). Strikingly, pWUR790 is linearized when incubated with PfAgo in absence of guides (Figure 5B). We observed this suspected guide-free PfAgo-mediated cleavage of pWUR790 in buffer with 250 mM NaCl, but not in buffer with 500 mM NaCl (Figure 5B). These findings suggest either that PfAgo cleaves plasmids independently of co-purified DNAs or that DNA guides are utilized but the concentration of such co-purified DNA is below the detection limit of the assay. It has previously been demonstrated that in vivo, TtAgo acquires guides that allow for targeting its expression vector (11). To rule out that this PfAgo activity was guided by co-purified RNA or by co-purified DNA guides that we were unable to detect, we used pWUR704 as target plasmid (Figure 5C). pWUR704 has minimal sequence similarity to the PfAgo expression vector (pWUR790; no sequences longer than 13 consecutive identical base pairs between the two plasmids). After incubation without PfAgo, the plasmid is present both in open circular and supercoiled configuration (Figure 5E, lane 1). When PfAgo is added, supercoiled pWUR704 is linearized even in absence of guides (Figure 5E, lane 2). Like pWUR790 cleavage, this activity is more pronounced at 250 mM NaCl compared to 500 mM NaCl. In contrast, TtAgo, which co-purified with detectable levels of siDNA, was unable to cleave pWUR704, unless synthetic siDNAs targeting pWUR790 were added (3). Low levels of guide-free PfAgo-mediated nicking of pWUR704 takes place within 1 h incubation at 75°C (Supplementary Figure S4). As PfAgo does not mediate RNA-guided activity within the same time span (Figure 4), it is unlikely that the activity on plasmid DNA is mediated by RNA that co-purified with PfAgo. Instead, these findings suggest that PfAgo has the potential to cleave the plasmid independently of guides.

Bottom Line: PfAgo utilizes small 5'-phosphorylated DNA guides to cleave both single stranded and double stranded DNA targets, and does not utilize RNA as guide or target.Thus, with respect to function and specificity, the archaeal PfAgo resembles bacterial Argonautes much more than eukaryotic Argonautes.These findings demonstrate that the role of Argonautes is conserved through the bacterial and archaeal domains of life and suggests that eukaryotic Argonautes are derived from DNA-guided DNA-interfering host defense systems.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Microbiology, Department of Agrotechnology and Food Sciences, Wageningen University, 6703 HB Wageningen, The Netherlands.

Show MeSH
Related in: MedlinePlus