Biology subjects

Kulbachiskiy, A.

Publications and source records attributed to Kulbachiskiy, A..

4 recordsLinked to original sources

bioRxiv2022

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 roseae (LrAgo) act as DNA-guided DNA nucleases in Escherichia coli and aid cell division in the presence of the gyrase inhibitor ciprofloxacin. Both pAgos are preferentially loaded with small DNA guides derived from the sites of replication termination. The amount of pAgo-associated small DNAs (smDNAs) from the termination sites is increased in the presence ciprofloxacin, suggesting that smDNA biogenesis depends on DNA replication and is stimulated by gyrase inhibition. Ciprofloxacin also 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 help to complete replication of chromosomal DNA by targeting the sites of termination, and may switch their functional activities when expressed in different host species.

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↗