Prokaryotic Argonaute nuclease cooperates with co-encoded RNase to acquire guide RNAs and target invader DNA.

Argonautes are an evolutionary conserved family of programmable nucleases that identify target nucleic acids using small guide oligonucleotides. In contrast to eukaryotic Argonautes (eAgos) that act on RNA, most studied prokaryotic Argonautes (pAgos) recognize DNA targets. Similarly to eAgos, pAgos can protect prokaryotic cells from invaders, but the biogenesis of guide oligonucleotides that confer them specificity to their targets remains poorly understood. Here, we have identified a new group of RNA-guided pAgo nucleases and demonstrated that a representative pAgo from this group, AmAgo from the mesophilic bacterium Alteromonas macleodii, binds guide RNAs of varying lengths for specific DNA targeting. Unlike most pAgos and eAgos, AmAgo is strictly specific to hydroxylated RNA guides containing a 5'-adenosine. AmAgo and related pAgos are co-encoded with a conserved RNA endonuclease from the HEPN superfamily (Ago-associated protein, Agap-HEPN). In vitro, Agap cleaves RNA between guanine and adenine nucleotides producing hydroxylated 5'-A guide oligonucleotides bound by AmAgo. In vivo, Agap cooperates with AmAgo in acquiring guide RNAs and counteracting bacteriophage infection. The AmAgo-Agap pair represents the first example of a pAgo system that autonomously produces RNA guides for DNA targeting and antiviral defense, which holds promise for programmable DNA targeting in biotechnology.

Multi-layered genome defences in bacteria.

Bacteria have evolved a variety of defence mechanisms to protect against mobile genetic elements, including restriction-modification systems and CRISPR-Cas. In recent years, dozens of previously unknown defence systems (DSs) have been discovered. Notably, diverse DSs often coexist within the same genome, and some co-occur at frequencies significantly higher than would be expected by chance, implying potential synergistic interactions. Recent studies have provided evidence of defence mechanisms that enhance or complement one another. Here, we review the interactions between DSs at the mechanistic, regulatory, ecological and evolutionary levels.

Sensing of DNA modifications by pAgo proteins in vitro.

Many prokaryotic Argonaute (pAgo) proteins act as programmable nucleases that use small guide DNAs for recognition and cleavage of complementary target DNA. Recent studies suggested that pAgos participate in cell defense against invader DNA and may also be involved in other genetic processes, including DNA replication and repair. The ability of pAgos to recognize specific targets potentially make them an invaluable tool for DNA manipulations. Here, we demonstrate that DNA-guided DNA-targeting pAgo nucleases from three bacterial species, DloAgo from Dorea longicatena, CbAgo from Clostridium butyricum and KmAgo from Kurthia massiliensis, can sense site-specific modifications in the target DNA, including 8-oxoguanine, thymine glycol, ethenoadenine and pyrimidine dimers. The effects of DNA modifications on the activity of pAgos strongly depend on their positions relative to the site of cleavage and are comparable to or exceed the effects of guide-target mismatches at corresponding positions. For all tested pAgos, the strongest effects are observed when DNA lesions are located at the cleavage position. The results demonstrate that DNA cleavage by pAgos is strongly affected by DNA modifications, thus making possible their use as sensors of DNA damage.

Bacterial Argonaute Proteins Aid Cell Division in the Presence of Topoisomerase Inhibitors in Escherichia coli.

Prokaryotic Argonaute (pAgo) proteins are guide-dependent nucleases that function in host defense against invaders. Recently, it was shown that TtAgo from Thermus thermophilus also participates in the completion of DNA replication by decatenating chromosomal DNA. Here, we show that two pAgos from cyanobacteria Synechococcus elongatus (SeAgo) and Limnothrix rosea (LrAgo) are active in heterologous Escherichia coli and aid cell division in the presence of the gyrase inhibitor ciprofloxacin, depending on the host double-strand break repair machinery. Both pAgos are preferentially loaded with small guide DNAs (smDNAs) derived from the sites of replication termination. Ciprofloxacin increases the amounts of smDNAs from the termination region and from the sites of genomic DNA cleavage by gyrase, suggesting that smDNA biogenesis depends on DNA replication and is stimulated by gyrase inhibition. Ciprofloxacin enhances asymmetry in the distribution of smDNAs around Chi sites, indicating that it induces double-strand breaks that serve as a source of smDNA during their processing by RecBCD. While active in E. coli, SeAgo does not protect its native host S. elongatus from ciprofloxacin. These results suggest that pAgo nucleases may help to complete replication of chromosomal DNA by promoting chromosome decatenation or participating in the processing of gyrase cleavage sites, and may switch their functional activities depending on the host species. IMPORTANCE Prokaryotic Argonautes (pAgos) are programmable nucleases with incompletely understood functions in vivo. In contrast to eukaryotic Argonautes, most studied pAgos recognize DNA targets. Recent studies suggested that pAgos can protect bacteria from invader DNA and counteract phage infection and may also have other functions including possible roles in DNA replication, repair, and gene regulation. Here, we have demonstrated that two cyanobacterial pAgos, SeAgo and LrAgo, can assist DNA replication and facilitate cell division in the presence of topoisomerase inhibitors in Escherichia coli. They are specifically loaded with small guide DNAs from the region of replication termination and protect the cells from the action of the gyrase inhibitor ciprofloxacin, suggesting that they help to complete DNA replication and/or repair gyrase-induced breaks. The results show that pAgo proteins may serve as a backup to topoisomerases under conditions unfavorable for DNA replication and may modulate the resistance of host bacterial strains to antibiotics.

Bacterial Argonaute nucleases reveal different modes of DNA targeting in vitro and in vivo.

Prokaryotic Argonaute proteins (pAgos) are homologs of eukaryotic Argonautes (eAgos) and are also thought to play a role in cell defense against invaders. However, pAgos are much more diverse than eAgos and little is known about their functional activities and target specificities in vivo. Here, we describe five pAgos from mesophilic bacteria that act as programmable DNA endonucleases and analyze their ability to target chromosomal and invader DNA. In vitro, the analyzed proteins use small guide DNAs for precise cleavage of single-stranded DNA at a wide range of temperatures. Upon their expression in Escherichia coli, all five pAgos are loaded with small DNAs preferentially produced from plasmids and chromosomal regions of replication termination. One of the tested pAgos, EmaAgo from Exiguobacterium marinum, can induce DNA interference between homologous sequences resulting in targeted processing of multicopy plasmid and genomic elements. EmaAgo also protects bacteria from bacteriophage infection, by loading phage-derived guide DNAs and decreasing phage DNA content and phage titers. Thus, the ability of pAgos to target multicopy elements may be crucial for their protective function. The wide spectrum of pAgo activities suggests that they may have diverse functions in vivo and paves the way for their use in biotechnology.

Programmable RNA targeting by bacterial Argonaute nucleases with unconventional guide binding and cleavage specificity.

Argonaute proteins are programmable nucleases that have defense and regulatory functions in both eukaryotes and prokaryotes. All known prokaryotic Argonautes (pAgos) characterized so far act on DNA targets. Here, we describe a new class of pAgos that uniquely use DNA guides to process RNA targets. The biochemical and structural analysis of Pseudooceanicola lipolyticus pAgo (PliAgo) reveals an unusual organization of the guide binding pocket that does not rely on divalent cations and the canonical set of contacts for 5'-end interactions. Unconventional interactions of PliAgo with the 5'-phosphate of guide DNA define its new position within pAgo and shift the site of target RNA cleavage in comparison with known Argonautes. The specificity for RNA over DNA is defined by ribonucleotide residues at the cleavage site. The analysed pAgos sense mismatches and modifications in the RNA target. The results broaden our understanding of prokaryotic defense systems and extend the spectrum of programmable nucleases with potential use in RNA technology.

RNA polymerase pausing, stalling and bypass during transcription of damaged DNA: from molecular basis to functional consequences.

Cellular DNA is continuously transcribed into RNA by multisubunit RNA polymerases (RNAPs). The continuity of transcription can be disrupted by DNA lesions that arise from the activities of cellular enzymes, reactions with endogenous and exogenous chemicals or irradiation. Here, we review available data on translesion RNA synthesis by multisubunit RNAPs from various domains of life, define common principles and variations in DNA damage sensing by RNAP, and consider existing controversies in the field of translesion transcription. Depending on the type of DNA lesion, it may be correctly bypassed by RNAP, or lead to transcriptional mutagenesis, or result in transcription stalling. Various lesions can affect the loading of the templating base into the active site of RNAP, or interfere with nucleotide binding and incorporation into RNA, or impair RNAP translocation. Stalled RNAP acts as a sensor of DNA damage during transcription-coupled repair. The outcome of DNA lesion recognition by RNAP depends on the interplay between multiple transcription and repair factors, which can stimulate RNAP bypass or increase RNAP stalling, and plays the central role in maintaining the DNA integrity. Unveiling the mechanisms of translesion transcription in various systems is thus instrumental for understanding molecular pathways underlying gene regulation and genome stability.

Role of the trigger loop in translesion RNA synthesis by bacterial RNA polymerase.

DNA lesions can severely compromise transcription and block RNA synthesis by RNA polymerase (RNAP), leading to subsequent recruitment of DNA repair factors to the stalled transcription complex. Recent structural studies have uncovered molecular interactions of several DNA lesions within the transcription elongation complex. However, little is known about the role of key elements of the RNAP active site in translesion transcription. Here, using recombinantly expressed proteins, in vitro transcription, kinetic analyses, and in vivo cell viability assays, we report that point amino acid substitutions in the trigger loop, a flexible element of the active site involved in nucleotide addition, can stimulate translesion RNA synthesis by Escherichia coli RNAP without altering the fidelity of nucleotide incorporation. We show that these substitutions also decrease transcriptional pausing and strongly affect the nucleotide addition cycle of RNAP by increasing the rate of nucleotide addition but also decreasing the rate of translocation. The secondary channel factors DksA and GreA modulated translesion transcription by RNAP, depending on changes in the trigger loop structure. We observed that although the mutant RNAPs stimulate translesion synthesis, their expression is toxic in vivo, especially under stress conditions. We conclude that the efficiency of translesion transcription can be significantly modulated by mutations affecting the conformational dynamics of the active site of RNAP, with potential effects on cellular stress responses and survival.

Four paralogous Gfh factors in the extremophilic bacterium Deinococcus peraridilitoris have distinct effects on various steps of transcription.

Gre factors are ubiquitous transcription regulators that stimulate co-transcriptional RNA cleavage by bacterial RNA polymerase (RNAP). Here, we show that the stress-resistant bacterium Deinococcus peraridilitoris encodes four Gre factor homologs, Gfh proteins, that have distinct effects on transcription by RNAP. Two of the factors, Gfh1α and Gfh2ß inhibit transcription initiation, and one of them, Gfh1α can also regulate transcription elongation. We show that this factor strongly stimulates transcriptional pausing and intrinsic termination in the presence of manganese ions but has no effect on RNA cleavage. Thus, some Gfh factors encoded by Deinococci serve as lineage-specific transcription inhibitors that may play a role in stress resistance, while the functions of others remain to be discovered.

Gre-family factors modulate DNA damage sensing by Deinococcus radiodurans RNA polymerase.

Deinococcus radiodurans is a highly stress resistant bacterium that encodes universal as well as lineage-specific factors involved in DNA transcription and repair. However, the effects of DNA lesions on RNA synthesis by D. radiodurans RNA polymerase (RNAP) have never been studied. We investigated the ability of this RNAP to transcribe damaged DNA templates and demonstrated that various lesions significantly affect the efficiency and fidelity of RNA synthesis. DNA modifications that disrupt correct base-pairing can strongly inhibit transcription and increase nucleotide misincorporation by D. radiodurans RNAP. The universal transcription factor GreA and Deinococcus-specific factor Gfh1 stimulate RNAP stalling at various DNA lesions, depending on the type of the lesion and the presence of Mn2+ ions, abundant divalent cations in D. radiodurans. Furthermore, Gfh1 stimulates the action of the Mfd translocase, which removes transcription elongation complexes paused at the sites of DNA lesions. Thus, Gre-family factors in D. radiodurans might have evolved to increase the efficiency of DNA damage recognition by the transcription and repair machineries in this bacterium.

Distinct effects of DNA lesions on RNA synthesis by Escherichia coli RNA polymerase.

DNA lesions can severely compromise genome stability and lead to cell death if unrepaired. RNA polymerase (RNAP) is known to serve as a sensor of DNA damage and to attract DNA repair factors to the damaged template sites. Here, we systematically investigated the ability of Escherichia coli RNAP to transcribe DNA templates containing various types of DNA lesions, and analyzed their effects on transcription fidelity. We showed that transcription is strongly inhibited on templates containing cyclobutane thymine dimers, 1,N6-ethenoadenine and abasic sites, while 8-oxoguanine and thymine glycol have mild effects on transcription efficiency. Similarly to many polymerases, E. coli RNAP follows the "A" rule during nucleotide insertion opposite abasic sites and bulky lesions, and can also incorporate and efficiently extend an adenine nucleotide opposite 8-oxoguanine. Mutations in RNAP regions around the templating nucleotide decrease the efficiency of translesion synthesis, likely by altering the RNAP-template contacts in the active site. Thus, DNA lesions can lead to distinct outcomes in transcription, depending on the severity of the damage and contacts of the damaged template with the active site of RNAP.

Gfh factors and NusA cooperate to stimulate transcriptional pausing and termination.

Lineage-specific Gfh factors from the radioresistant bacterium Deinococcus radiodurans, which bind within the secondary channel of RNA polymerase, stimulate transcriptional pausing at a wide range of pause signals (elemental, hairpin-dependent, post-translocated, backtracking-dependent, and consensus pauses) and increase intrinsic termination. Universal bacterial factor NusA, which binds near the RNA exit channel, enhances the effects of Gfh factors on termination and hairpin-dependent pausing but do not act on other pause sites. It is proposed that NusA and Gfh target different steps in the pausing pathway and may act together to regulate transcription under stress conditions. Thus, transcription factors that interact with nascent RNA in the RNA exit channel can communicate with secondary channel regulators to modulate RNA polymerase activities.

Regulation of transcription initiation by Gfh factors from Deinococcus radiodurans.

Transcription factors of the Gre family bind within the secondary channel of bacterial RNA polymerase (RNAP) directly modulating its catalytic activities. Universally conserved Gre factors activate RNA cleavage by RNAP, by chelating catalytic metal ions in the RNAP active site, and facilitate both promoter escape and transcription elongation. Gfh factors are Deinococcus/Thermus-specific homologues of Gre factors whose transcription functions remain poorly understood. Recently, we found that Gfh1 and Gfh2 proteins from Deinococcus radiodurans dramatically stimulate RNAP pausing during transcription elongation in the presence of Mn2+, but not Mg2+, ions. In contrast, we show that Gfh1 and Gfh2 moderately inhibit transcription initiation in the presence of either Mg2+ or Mn2+ ions. By using a molecular beacon assay, we demonstrate that Gfh1 and Gfh2 do not significantly change promoter complex stability or the rate of promoter escape by D. radiodurans RNAP. At the same time, Gfh factors significantly increase the apparent KM value for the 5'-initiating nucleotide, without having major effects on the affinity of metal ions for the RNAP active site. Similar inhibitory effects of Gfh factors are observed for transcription initiation on promoters recognized by the principal and an alternative σ factor. In summary, our data suggest that D. radiodurans Gfh factors impair the binding of initiating substrates independently of the metal ions bound in the RNAP active site, but have only mild overall effects on transcription initiation. Thus the mechanisms of modulation of RNAP activity by these factors are different for various steps of transcription.

Regulation of transcriptional pausing through the secondary channel of RNA polymerase.

Transcriptional pausing has emerged as an essential mechanism of genetic regulation in both bacteria and eukaryotes, where it serves to coordinate transcription with other cellular processes and to activate or halt gene expression rapidly in response to external stimuli. Deinococcus radiodurans, a highly radioresistant and stress-resistant bacterium, encodes three members of the Gre family of transcription factors: GreA and two Gre factor homologs, Gfh1 and Gfh2. Whereas GreA is a universal bacterial factor that stimulates RNA cleavage by RNA polymerase (RNAP), the functions of lineage-specific Gfh proteins remain unknown. Here, we demonstrate that these proteins, which bind within the RNAP secondary channel, strongly enhance site-specific transcriptional pausing and intrinsic termination. Uniquely, the pause-stimulatory activity of Gfh proteins depends on the nature of divalent ions (Mg(2+) or Mn(2+)) present in the reaction and is also modulated by the nascent RNA structure and the trigger loop in the RNAP active site. Our data reveal remarkable plasticity of the RNAP active site in response to various regulatory stimuli and highlight functional diversity of transcription factors that bind inside the secondary channel of RNAP.