Biology subjects

Esyunina, D.

Publications and source records attributed to Esyunina, D..

6 recordsLinked to original sources

bioRxiv2023

DNA-targeting short Argonaute triggers effector nuclease to protect bacteria from invaders

Two prokaryotic defence systems, Argonautes (pAgos) and CRISPR-Cas, detect invader nucleic acids using complementary guides. Upon recognition, the target is cleaved through nuclease activities of pAgo or Cas proteins thus protecting the cell from invasion. However, not all pAgos are active nucleases. Members of a large clade of short pAgos bind nucleic acid guides but lack nuclease activity suggesting a different mechanism of action. Here, we have investigated short pAgo from Novosphingopyxis baekryungensis (NbaAgo). We have shown that NbaAgo forms a heterodimeric complex, SPARDA, with a co-encoded effector nuclease. RNA-guided target DNA recognition unleashes the nuclease activity of SPARDA leading to indiscriminate collateral cleavage of DNA and RNA. Activation of SPARDA results in cell death during plasmid transformation or phage infection, thus protecting bacterial population from invaders. The collateral activity of SPARDA allows highly sensitive detection of specific DNA targets. SPARDA expands the list of prokaryotic immune systems that elicit suicidal cell response with a unique range of nuclease activities, creating additional opportunities for biotechnologies.

molecular biology↗

bioRxiv2022

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

Prokaryotic Argonaute proteins (pAgos) are homologs of eukaryotic Argonautes (eAgos) that were similarly proposed to play a role in cell defense against invaders. However, pAgos are much more diverse than eAgos and very little is known about their functional activity and target specificity in vivo. Here, we describe five pAgo proteins from mesophilic bacteria that act as DNA-guided 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 plasmid DNA and from chromosomal regions of replication termination. One of the tested pAgos, EmaAgo from Exiguobacterium marinum can induce DNA interference between multicopy sequences resulting in targeted processing of homologous plasmid and chromosomal loci. EmaAgo also protects bacteria from bacteriophage infection and is preferentially loaded with phage guide DNAs suggesting that 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.

molecular biology↗

bioRxiv2022

Specific targeting of plasmids with Argonaute enables genome editing

Prokaryotic Argonautes (pAgos) are programmable nucleases involved in cell defense against invading DNA. Recent studies showed that pAgos can bind small single-stranded guide DNAs (gDNAs) to recognize and cleave complementary DNA in vitro. In vivo pAgos preferentially target plasmids, phages and multicopy genetic elements. Here, we reveal that CbAgo nuclease from Clostridium butyricum can be used for genomic DNA cleavage and engineering in bacteria. CbAgo-dependent targeting of genomic loci with plasmid-derived gDNAs promotes recombination between plasmid and chromosomal DNA. Efficient genome cleavage and recombineering depends on the catalytic activity of CbAgo, its interactions with gDNAs, and the extent of homology between plasmid and chromosomal sequences. Specific targeting of plasmids with Argonautes can be used to integrate plasmid-encoded sequences into the chromosome thus enabling genome editing. One-Sentence SummaryProkaryotic Argonaute nuclease induces DNA interference between plasmid and chromosomal DNA to promote genome recombineering.

molecular biology↗

bioRxiv2021

A programmable pAgo nuclease with universal guide and target specificity from the mesophilic bacterium Kurthia massiliensis

Argonaute proteins are programmable nucleases that are found in both eukaryotes and prokaryotes and provide defense against invading genetic elements. Although some prokaryotic Argonautes (pAgos) were shown to recognize RNA targets in vitro, the majority of studied pAgos have strict specificity toward DNA, which limits their practical use in RNA-centric applications. Here, we describe a unique KmAgo nuclease from the mesophilic bacterium Kurthia massiliensis that can be programmed with either DNA or RNA guides and can precisely cleave both DNA and RNA targets. KmAgo preferentially binds 16-20 nt long 5'-phosphorylated guide molecules with no strict specificity for their sequence and is active in a wide range of temperatures. In bacterial cells, KmAgo is loaded with small DNAs with no obvious sequence preferences suggesting that it can uniformly target genomic sequences. Target cleavage by KmAgo depends on the formation of secondary structure indicating that KmAgo can be used for structural probing of RNA targets. Mismatches between the guide and target sequences greatly affect the efficiency and precision of target cleavage, depending on the mismatch position and the nature of the reacting nucleic acid. These properties of KmAgo open the way for its use for highly specific nucleic acid detection and cleavage.

molecular biology↗

bioRxiv2020

Structural basis of ribosomal RNA transcription regulation

Ribosomal RNA (rRNA) is the most highly expressed gene in rapidly growing bacteria and is drastically downregulated under stress conditions by the global transcriptional regulator DksA and the alarmone ppGpp. To reveal the mechanism of highly regulated rRNA transcription, we determined cryo-electron microscopy structures of the Escherichia coli RNA polymerase (RNAP) {sigma}70 holoenzyme at different steps of rRNA promoter recognition with and without DksA/ppGpp. RNAP contacts the UP element of rRNA promoter using the dimerized subunit carboxyl-terminal domain and scrunches the template DNA with the {sigma}finger and {beta}lid to select a transcription start site favorable for rRNA expression. Promoter DNA binding to RNAP induces conformational change of the {sigma} domain 2 that opens a gate for DNA loading and ejects {sigma}1.1 from the RNAP cleft to facilitate open complex formation. DksA/ppGpp binding to RNAP also opens the DNA loading gate, but it is not coupled to {sigma}1.1 ejection and impedes the open complex formation of the rRNA promoter due to its G+C rich discriminator sequence. Mutations in {sigma}1.1 or the {beta}lid stabilize the RNAP and rRNA promoter complex and decrease its sensitivity to DksA/ppGpp. These results provide a molecular basis for exceptionally active rRNA transcription and for its vulnerability to DksA/ppGpp.

molecular biology↗

bioRxiv2020

pAgo-induced DNA interference protects bacteria from invader DNA

Members of the conserved Argonaute protein family use small RNA guides to find their mRNA targets to regulate gene expression and suppress mobile genetic elements in eukaryotes. Argonautes are also present in many bacterial and archaeal species 1-3. Unlike eukaryotic proteins, several studied prokaryotic Argonautes use small DNA guides to cleave DNA, a process dubbed DNA interference 4-8. However, the natural functions and targets of DNA interference are poorly understood and the mechanisms of DNA guide generation and target discrimination remain unknown. Here, we studied the in vivo activities of a bacterial Argonaute nuclease CbAgo and demonstrated that it induces cleavage of multicopy genetic elements, including plasmids, transposons and repetitive chromosomal loci. Generation of small DNA guides employed by CbAgo requires cooperation between its intrinsic endonuclease activity and the cellular double-strand break repair machinery. The mechanism of guide generation ensures that small DNA guides are enriched in sequences that target foreign DNA and endows CbAgo with capacity to eliminate plasmids and fight phage infection. Similar principles may underlie the specificity of self-nonself discrimination by diverse defense systems in prokaryotes.

molecular biology↗