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. 2020 May;17(5):677-688.
doi: 10.1080/15476286.2020.1724716. Epub 2020 Feb 16.

Genome-wide DNA sampling by Ago nuclease from the cyanobacterium Synechococcus elongatus

Anna Olina  1 , Anton Kuzmenko  1 , Maria Ninova  2 , Alexei A Aravin  2 , Andrey Kulbachinskiy  1 , Daria Esyunina  1
Affiliations

Genome-wide DNA sampling by Ago nuclease from the cyanobacterium Synechococcus elongatus

Anna Olina et al. RNA Biol. 2020 May.

Abstract

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.

Keywords: Argonaute; SeAgo; Synechococcus elongatus; ori and ter sites; programmable DNA nuclease.

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Figures

Figure 1.
Figure 1.
Site-specific ssDNA cleavage by SeAgo. (A) Structural model of SeAgo. The N-, PAZ, MID, and PIWI domains are shown in yellow, red, green and blue, respectively. The modelling was performed using the Phyre2 web tool [19], based on the structure of TtAgo from T. thermophilus (PDB 2ETN [11]) as the template. (B) Structure of TtAgo (4NCB [8]). (C) Scheme of the guide and target DNAs used in the cleavage assay (see Table S1 for complete oligonucleotide sequences). (D) Kinetics of ssDNA cleavage by SeAgo in the presence of Mg2+ and Mn2+. The reaction products were analysed by denaturing urea PAGE followed by SYBR Gold staining. The cleavage site is located in the middle of the 50 nt target oligonucleotide, which also contains a 3ʹ-Cy3 fluorophore (T-tDNA_Cy3, Table S1), so that the resulting 25 nt 3ʹ-fragment has lower mobility in the gel than the 5ʹ-fragment (see Fig. S3). (E) Effects of mismatches in different guide parts on target DNA cleavage by SeAgo. The positions and the identities of the mismatches are indicated above the gel. (F) Quantification of the cleavage efficiencies for guide DNAs containing mismatches at various positions relative to the control fully complementary guide oligonucleotide (fc, fold-change). Means and standard deviations from three independent experiments are shown (* – p < 0.05; ** – p < 0.01). (G) Effect of SeSSB on ssDNA cleavage by SeAgo. The experiments were performed with 500 nM SeAgo, 500 nM guide DNA, 100 nM target DNA and 400 nM SeSSB in Mn2+-containing buffer. Positions of the target (T), guide (G) and product (P) DNAs are indicated.
Figure 2.
Figure 2.
Plasmid DNA processing by SeAgo. (A) Scheme of the target plasmid (pJET_target). Positions of guide DNAs, promoter (T7A1) and terminator (T) regions are indicated. The predicted cleavage sites are shown with arrowheads. (B) Analysis of plasmid processing by nondenaturing agarose gel-electrophoresis. The plasmid was incubated with empty SeAgo or guide-loaded SeAgo in the absence or in the presence of SeSSB. (C) Scheme of an experiment aimed at the analysis of a possible role of transcription in DNA processing by SeAgo. (D) Analysis of the effect of transcription on plasmid processing. The reactions were performed either in the absence or in the presence of RNA polymerase and NTPs. SeAgo, guide DNA and plasmid DNA were present at 500 nM, 500 nM and 2 nM, respectively. T, guide DNA corresponding to the transcribed DNA strand in the target region; NT, guide DNA corresponding to the nontranscribed DNA strand; SC, supercoiled plasmid; OC, open-circle relaxed plasmid; Lin, linearized plasmid.
Figure 3.
Figure 3.
Expression of SeAgo in S. elongatus. (A) Schematics of the S. elongatus strains used for in vivo analysis. Positions of the chromosomal pAgo gene (replaced with luciferase in the ΔAgo strain), spectinomycin resistance gene, His6-tagged wild-type or catalytically inactive SeAgo, and corresponding promoter regions are indicated. (B) Comparison of the growth curves for the wild-type and ΔAgo (luciferase) strains, with an indication of time points used for the analysis of SeAgo expression. (C) Measurements of the SeAgo expression in the wild-type strain of S. elongatus by RT-qPCR. The mRNA levels are normalized to the rnpB housekeeping gene and are shown relative to the expression level at the 4th day of cell growth. (D) Analysis of the activity of the luciferase gene placed under control of the SeAgo promoter in the ΔAgo strain. The luminescence levels were normalized by the amounts of total protein in the samples after cell lysis. (E) Comparison of the growth curves for various strains of S. elongatus analysed in this study.
Figure 4.
Figure 4.
Analysis of small DNAs associated with SeAgo in vivo. (A) Isolation of small nucleic acids bound to SeAgo during its expression in S. elongatus (left) or E. coli (right). The experiments were performed with His6-tagged chromosomally encoded SeAgo in S. elongatus (↑AgoWT strain) or plasmid-encoded SeAgo in E. coli. After cell lysis, SeAgo was purified by Co2+-affinity chromatography, associated nucleic acids were (or were not) treated with alkaline phosphatase, labelled using γ-32P-ATP and polynucleotide kinase (PNK, radioactive labelling is indicated with asterisks), and treated with DNase I or RNaseA, as indicated on the figure, followed by denaturing PAGE. DNA markers are shown on the right of each gel. (B) Length distribution of SeAgo-associated small DNAs in sequenced libraries. (C) Sequence logos for small DNAs aligned by their 5ʹ-ends; only reads ≥16 nt were used for this analysis. (D) Distribution of small DNAs along with the endogenous pANL plasmid. The small DNA coverage is plotted as rpkm (reads per kilobase per million reads in the small DNA library; open orange circles) over the plasmid coordinate. The blue line shows local polynomial regression fitting (LOESS smoothing). The predicted origin of replication is shown with a two-way red arrow [31]. (E) Distribution of small DNAs along the S. elongatus genome. Genomic positions are indicated on the x-axis. The rpkm values were averaged over 5 kb windows. The three major peaks of small DNAs and predicted positions of the replication origin and termination regions are indicated.
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This work was supported by the Russian Science Foundation [16-14-10377];Russian Foundation for Basic Research [18-29-07086].

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