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Guide-independent DNA cleavage by archaeal Argonaute from
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Methanocaldococcus jannaschii
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Adrian Zander
1,#
, Sarah Willkomm
1,#
, Sapir Ofer
2
, Marleen van Wolferen
3
, Luisa
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Egert
1
, Sabine Buchmeier
4
, Sarah Stöckl
1
, Philip Tinnefeld
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, Sabine Schneider
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,
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Andreas Klingl
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, Sonja-Verena Albers
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, Finn Werner
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, Dina Grohmann
1,*
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1
Department of Microbiology & Archaea Centre, University of Regensburg,
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Regensburg, 93053, Germany
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2
Institute for Structural and Molecular Biology, Division of Biosciences, University
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College London, London WC1E 6BT, United Kingdom
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3
Molecular Biology of Archaea, Institute of Biology II,
University of Freiburg,
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Microbiology, Schaenzlestraße 1, 79104 Freiburg, Germany
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4
Institute of Physical and Theoretical Chemistry – NanoBioSciences, Technische
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Universität Braunschweig-BRICS, Rebenring 56, 38106 Braunschweig, Germany
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5
Center for Integrated Protein Science Munich CIPSM, Department of Chemistry,
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Technische Universität München, Lichtenbergstrasse 4, 85748 Garching, Germany
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6
Biocentre of the LMU Munich, Department Biology I – Plant Development,
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Großhadernerstr. 2-4, 82152 Planegg-Martinstried, Germany
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#
These authors contributed equally
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*For correspondence: dina.grohmann@ur.de (DG)
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Keywords: Argonaute, archaea, DNA-guided gene silencing
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Abstract
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Prokaryotic Argonaute proteins acquire guide strands derived from invading or
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mobile genetic elements via an unknown pathway to direct guide-dependent
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cleavage of foreign DNA. Here, we report that Argonaute from the archaeal
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organism Methanocaldococcus jannaschii (MjAgo) possesses two modes of action:
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the canonical guide-dependent endonuclease activity and a non-guided DNA
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endonuclease activity. The latter allows MjAgo to process long double stranded
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DNAs, including circular plasmid DNAs and genomic DNAs. Degradation of substrates
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in a guide-independent fashion primes MjAgo for subsequent rounds of DNA
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cleavage. Chromatinised genomic DNA is resistant to MjAgo degradation and
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recombinant histones protect DNA from cleavage in vitro. Mutational analysis shows
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that key residues important for guide-dependent target processing are also involved
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in guide-independent MjAgo function. This is the first-time characterisation of a
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guide-independent cleavage activity for an Argonaute protein potentially serving as
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guide biogenesis pathway in a prokaryotic system.
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Introduction
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Argonaute (Ago) proteins are crucially involved in RNA-guided or DNA-guided
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degradation of target nucleic acids.
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Present in all three domains of life, they bind
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guide strands in vivo to target complementary nucleic acids. Eukaryotic Agos interact
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with cytoplasmic RNA substrates 18–23 bp in length
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while prokaryotic Agos
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(pAgos) bind and process a variety of DNA and RNA substrates.
7-11
Among them,
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Agos from the archaeal organisms Methanocaldococcus jannaschii (MjAgo),
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Pyrococcus furiosus (PfAgo) and Natronobacterium gregoryi (NgAgo) are the only
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Ago variants that exclusively cleave DNA substrates using a DNA guide in vitro.
7,11-13
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Guide recognition is mediated by a phosphate group at the guide’s 5’-end, which is
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coordinated in the Mid domain by conserved amino-acid side chain interactions.
14-18
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One exception are the recently characterized bacterial Agos from Marinitoga
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piezophila (MpAgo) and Thermotoga profunda (TpAgo), which recognise RNA guides
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with a 5’-hydroxyl group.
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Loaded with the guide, Ago binds partially or fully
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complementary target nucleic acids via Watson-Crick base pairing. Only fully
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complementary target strands are cleaved by Ago. The catalytic site resides in the
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PIWI domain. Notably, numerous pAgos, especially short Argonaute variants, have
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an incomplete catalytic site rendering them inactive.
2
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While the structural organization of pAgos is well understood, their biological role is
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still not fully revealed. In vivo studies have only been reported for the bacterial
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organisms Thermus thermophilus (Tt) and Rhodobacter sphaeroides (Rs). In both
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cases, Ago appears to play a role in host defence.
8,20
TtAgo acquires guide DNAs 13-
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25 nt in length that carry the canonical 5’-phosphate. Overexpression of TtAgo in
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T.thermophilus leads to its association with DNA sequences mainly derived from the
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TtAgo expression plasmid.
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TtAgo, MpAgo and NgAgo cleave plasmids
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complementary to their guide DNA by nicking both strands of the plasmid DNA.
8,19,21
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In contrast, RsAgo is most probably involved in RNA-guided DNA silencing.
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The
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majority of sequences acquired by RsAgo map to genome-encoded foreign nucleic
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acids like transposons and phage genes.
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It was suggested that the catalytically
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inactive RsAgo acts in concert with a nuclease, which is encoded in the same operon,
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thereby mediating RNA-guided silencing in R. sphaeroides.
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In this study, we describe the guide-independent endonuclease activity of the
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archaeal MjAgo. We show that MjAgo can process long dsDNAs including plasmids
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and genomic DNA in a guide-independent manner, which leads to the generation of
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cleavage products potentially suitable as guides. Using these cleavage products in a
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second cleavage round accelerates processing of the original substrate DNA
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suggesting a priming mechanism. Only the chromatinised state of M.jannaschii’s
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genomic DNA is protected against MjAgo-mediated degradation, and histone
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proteins are likely to confer this protection. Additionally, our structure-based
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mutational analysis reveals amino acids and structural elements of crucial
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importance for the guide-independent cleavage activity of MjAgo. Taken together,
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our findings support a scenario in which the non-guided endonuclease activity of
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MjAgo represents a mechanism to protect a prokaryotic organism against foreign
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genetic elements.
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RESULTS
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MjAgo can utilize non-canonical DNA guides for cleavage of DNA targets
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First, we analysed the guide length tolerance of MjAgo. We tested 5’-phosphorylated
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DNA guides 13–23 nt in length in a guide-dependent target cleavage assay (Figure
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1a). Starting from a minimal guide length of 15 nt, MjAgo accepted all guide lengths
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up to 23 nt (Figure 1b). We also found efficient cleavage of a non-canonical
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substrate (41 nt guide / 41 nt target) even without a 5’-phosphate (Figure 1c,d and
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Supplementary Figure 1). None of the substrates was cleaved by the catalytic
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mutant MjAgo
E541A
(Supplementary Figure 2). Next, we tested whether MjAgo
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exhibits orientated loading and cleavage of the 41 nt guide/41 nt target substrate
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using target strands that either carry the fluorescent label at the 5’-end or towards
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the 3’-end together with guide strands with and without a 5’-phosphate group
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(Figure 1e). In case of a 5’-phosphorylated 41nt guide, the production of a canonical
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cleavage product was observed with cleavage occurring opposite nucleotide 10/11
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of the guide strand. However, the majority of the substrate is preferentially cleaved
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from the 5’-end of the target strand in a stepwise manner (Figure 1f). From a
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structural perspective, it is not feasible that both ends of a 41 nt long guide are
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accommodated in the Mid and PAZ domain indicating that MjAgo employs a non-
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canonical binding and cleavage mechanism.
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MjAgo cleaves long linear and circular double-stranded DNA in a guide-
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independent fashion
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Next, we tested significantly longer substrates and incubated MjAgo with a 750 bp
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dsDNA and circular double-stranded plasmid DNAs. In both cases, we found cleavage
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of the substrate in a guide-independent manner (the MjAgo
E541A
mutant did not
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process these DNAs) (Figure 2a,c,d). The DNA is gradually cleaved over time
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(Supplementary Figure 3) until final cleavage products smaller than 100 bp
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accumulate (Figure 2 and 3). EDTA prevents cleavage of long dsDNA by MjAgo
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(Supplementary Figure 4) suggesting that the conserved catalytic tetrad, which
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coordinates two metal ions, carries out the cleavage reaction. DNA degradation
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occurs quickly at physiological relevant high temperatures of 75°C-85°C (Figure 2d).
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