Biology subjects

Yudin, D.

Publications and source records attributed to Yudin, D..

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↗

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↗

bioRxiv2019

Programmable DNA cleavage by Ago nucleases from mesophilic bacteria Clostridium butyricum and Limnothrix rosea

Argonaute (Ago) proteins are the key players in RNA interference in eukaryotes, where they function as RNA-guided RNA endonucleases. Prokaryotic Argonautes (pAgos) are much more diverse than their eukaryotic counterparts but their cellular functions and mechanisms of action remain largely unknown. Some pAgos were shown to use small DNA guides for endonucleolytic cleave of complementary DNA in vitro. However, previously studied pAgos from thermophilic prokaryotes function at elevated temperatures which limits their potential use as a tool in genomic applications. Here, we describe two pAgos from mesophilic bacteria, Clostridium butyricum (CbAgo) and Limnothrix rosea (LrAgo), that act as DNA-guided DNA nucleases at physiological temperatures. In contrast to previously studied pAgos, CbAgo and LrAgo can use not only 5-phosphorylated but also 5-hydroxyl DNA guides, with diminished precision of target cleavage. Both LrAgo and CbAgo can tolerate guide/target mismatches in the seed region, but are sensitive to mismatches in the 3-guide region. CbAgo is highly active under a wide range of conditions and can be used for programmable endonucleolytic cleavage of both single-stranded and double-stranded DNA substrates at moderate temperatures. The biochemical characterization of mesophilic pAgo proteins paths the way for their use for DNA manipulations both in vitro and in vivo.

molecular biology↗