DNA breathing initiated strand displacement circuits for mimicking ant foraging with nanopore readout

DNA strand displacement reaction is essential for the development of molecular computing based on DNA nanotechnology. Additional DNA strand exchange strategies with high selectivity for input will enable novel complex systems including biosensing applications. Most approaches use bulk readout methods based on fluorescent probes that complicate the monitoring of parallel computations. Herein we propose an autocatalytic strand displacement (ACSD) circuit, which is initiated by DNA breathing and accelerated by seesaw catalytic reaction. The special initiation mechanism of the ACSD circuit enables detection of single base mutations at multiple sites in the input strand with much higher sensitivity than classic toehold-mediated strand displacement. A swarm intelligence model is constructed using the ACSD circuit to mimic foraging behaviour of ants. We introduce a multiplexed nanopore sensing platform to report the output results of a parallel path selection system on the single-molecule level. The ACSD strategy and nanopore multiplexed readout method enhance the toolbox for the future development of DNA computing.

Fission yeast Rad8/HLTF facilitates Rad52-dependent chromosomal rearrangements through PCNA lysine 107 ubiquitination

Gross chromosomal rearrangements (GCRs), including translocation, deletion, and inversion, can cause cell death and genetic diseases such as cancer in multicellular organisms. Rad51, a DNA strand exchange protein, suppresses GCRs by repairing spontaneous DNA damage through a conservative way of homologous recombination, gene conversion. On the other hand, Rad52 that catalyzes single-strand annealing (SSA) causes GCRs using homologous sequences. However, the detailed mechanism of Rad52-dependent GCRs remains unclear. Here, we provide genetic evidence that fission yeast Rad8/HLTF facilitates Rad52-dependent GCRs through the ubiquitination of lysine 107 (K107) of PCNA, a DNA sliding clamp. In rad51Δ cells, loss of Rad8 eliminated 75% of the isochromosomes resulting from centromere inverted repeat recombination, showing that Rad8 is essential for the formation of the majority of isochromosomes in rad51Δ cells. Rad8 HIRAN and RING finger mutations reduced GCRs, suggesting that Rad8 facilitates GCRs through 3’ DNA-end binding and ubiquitin ligase activity. Mms2 and Ubc4 but not Ubc13 ubiquitin-conjugating enzymes were required for GCRs. Consistent with this, mutating PCNA K107 rather than the well-studied PCNA K164 reduced GCRs. Rad8-dependent PCNA K107 ubiquitination facilitates Rad52-dependent GCRs, as PCNA K107R, rad8, and rad52 mutations epistatically reduced GCRs. In contrast to GCRs, PCNA K107R did not significantly change gene conversion rates, suggesting a specific role of PCNA K107 ubiquitination in GCRs. PCNA K107R enhanced temperature-sensitive growth defects of DNA ligase I cdc17-K42 mutant, implying that PCNA K107 ubiquitination occurs when Okazaki fragment maturation fails. Remarkably, K107 is located at the interface between PCNA subunits, and an interface mutation D150E bypassed the requirement of PCNA K107 and Rad8 ubiquitin ligase for GCRs. These data suggest that Rad8-dependent PCNA K107 ubiquitination facilitates Rad52-dependent GCRs by changing the PCNA clamp structure.

Control of the serine integrase reaction: roles of the coiled-coil and helix E regions in DNA site synapsis and recombination

Bacteriophage serine integrases catalyze highly specific recombination reactions between defined DNA segments called att sites. These reactions are reversible depending upon the presence of a second phage-encoded directionality factor. The bipartite C-terminal DNA binding region of integrases includes a recombinase domain (RD) connected to a zinc-binding domain (ZD), which contains a long flexible coiled-coil (CC) motif that extends away from the bound DNA. We directly show that the identities of the phage A118 integrase att sites are specified by the DNA spacing between the RD and ZD DNA recognition determinants, which in turn, directs the relative trajectories of the CC motifs on each subunit of the att -bound integrase dimer. Recombination between compatible dimer-bound att sites requires minimal length CC motifs and 14 residues surrounding the tip where pairing of CC motifs between synapsing dimers occurs. Our alanine-scanning data suggests that molecular interactions between CC motif tips may differ in integrative ( attP x attB ) and excisive ( attL x attR ) recombination reactions. We identify mutations in 5 residues within the integrase oligomerization helix that control the remodeling of dimers into tetramers during synaptic complex formation. Whereas most of these gain-of-function mutants still require the CC motifs for synapsis, one mutant efficiently, but indiscriminantly, forms synaptic complexes without the CC motifs. However, the CC motifs are still required for recombination, suggesting a function for the CC motifs after initial assembly of the integrase synaptic tetramer. Importance The robust and exquisitely-regulated site-specific recombination reactions promoted by serine integrases are integral to the life cycle of temperate bacteriophage, and in the case of the A118 prophage, are an important virulence factor by Listeria monocytogenes . The properties of these recombinases have led to their repurposing into tools for genetic engineering and synthetic biology. In this report, we identify determinants regulating synaptic complex formation between correct DNA sites, including the DNA architecture responsible for specifying the identity of recombination sites, features of the unique coiled-coil structure on the integrase that are required to initiate synapsis, and amino acid residues on the integrase oligomerization helix that control the remodeling of synapsing dimers into a tetramer active for DNA strand exchange.

THE DEAD-Box PROTEIN Csha IN STAPHYLOCOCCUS AUREUS CONTAINS ATP-INDEPENDENT DNA STRAND ANNEALING AND EXCHANGE ACTIVITIES

DEAD-box proteins (DBPs) that are usually RNA helicases have important roles in eukaryotic and bacterial RNA metabolism. Recent studies have reported that certain prokaryotic DBPs exhibit ATP-independent nucleic acid displacement and annealing activities. We investigated one putative RNA helicase, CshA DEAD-box protein, from vancomycin-resistant Staphylococcus aureus strain Mu 50 for ATP-independent activities on nucleic acids. We herein report that CshA has two novel ATP-independent activities - annealing of complementary single-stranded DNA (ssDNA) and strand exchange on short double-stranded DNA (dsDNA). These DNA strand annealing and exchange activities are independent of Mg2+ ion or ATP binding and hydrolysis. ssDNA annealing activity as well as versatile DNA strand exchange activity of CshA suggests a possible role in dsDNA break repair processes.

PprA Protein Inhibits DNA Strand Exchange and ATP Hydrolysis of Deinococcus RecA and Regulates the Recombination in Gamma-Irradiated Cells

DrRecA and PprA proteins function are crucial for the extraordinary resistance to γ-radiation and DNA strand break repair in Deinococcus radiodurans. DrRecA mediated homologous recombination help in DNA strand break repair and cell survival, while the PprA protein confers radio-resistance via its roles in DNA repair, genome maintenance, and cell division. Genetically recA and pprA genes interact and constitute an epistatic group however, the mechanism underlying their functional interaction is not clear. Here, we showed the physical and functional interaction of DrRecA and PprA protein both in solution and inside the cells. The absence of the pprA gene increases the recombination frequency in gamma-irradiated D. radiodurans cells and genomic instability in cells growing under normal conditions. PprA negatively regulates the DrRecA functions by inhibiting DrRecA mediated DNA strand exchange and ATPase function in vitro. Furthermore, it is shown that the inhibitory effect of PprA on DrRecA catalyzed DNA strand exchange was not due to sequestration of homologous dsDNA and was dependent on PprA oligomerization and DNA binding property. Together, results suggest that PprA is a new member of recombination mediator proteins (RMPs), and able to regulate the DrRecA function in γ-irradiated cells by protecting the D. radiodurans genome from hyper-recombination and associated negative effects.

Identification of a Selective RelA Inhibitor Based on DSE-FRET Screening Methods

Nuclear factor-κB (NF-κB) is an important transcription factor involved in various biological functions, including tumorigenesis. Hence, NF-κB has attracted attention as a target factor for cancer treatment, leading to the development of several inhibitors. However, existing NF-κB inhibitors do not discriminate between its subunits, namely, RelA, RelB, cRel, p50, and p52. Conventional methods used to evaluate interactions between transcription factors and DNA, such as electrophoretic mobility shift assay and luciferase assays, are unsuitable for high-throughput screening (HTS) and cannot distinguish NF-κB subunits. We developed a HTS method named DNA strand exchange fluorescence resonance energy transfer (DSE-FRET). This assay is suitable for HTS and can discriminate a NF-κB subunit. Using DSE-FRET, we searched for RelA-specific inhibitors and verified RelA inhibition for 32,955 compounds. The compound A55 (2-(3-carbamoyl-6-hydroxy-4-methyl-2-oxopyridin-1(2H)-yl) acetic acid) selectively inhibited RelA–DNA binding. We propose that A55 is a seed compound for RelA-specific inhibition and could be used in clinical applications.

Single-Molecule Insights into ATP-Dependent Conformational Dynamics of Nucleoprotein Filaments of Deinococcus radiodurans RecA

Deinococcus radiodurans (Dr) has one of the most robust DNA repair systems, which is capable of withstanding extreme doses of ionizing radiation and other sources of DNA damage. DrRecA, a central enzyme of recombinational DNA repair, is essential for extreme radioresistance. In the presence of ATP, DrRecA forms nucleoprotein filaments on DNA, similar to other bacterial RecA and eukaryotic DNA strand exchange proteins. However, DrRecA catalyzes DNA strand exchange in a unique reverse pathway. Here, we study the dynamics of DrRecA filaments formed on individual molecules of duplex and single-stranded DNA, and we follow conformational transitions triggered by ATP hydrolysis. Our results reveal that ATP hydrolysis promotes rapid DrRecA dissociation from duplex DNA, whereas on single-stranded DNA, DrRecA filaments interconvert between stretched and compressed conformations, which is a behavior shared by E. coli RecA and human Rad51. This indicates a high conservation of conformational switching in nucleoprotein filaments and suggests that additional factors might contribute to an inverse pathway of DrRecA strand exchange.

Two auxiliary factors promote Dmc1-driven DNA strand exchange via stepwise mechanisms

Homologous recombination (HR) is a universal mechanism operating in somatic and germ-line cells, where it contributes to the maintenance of genome stability and ensures the faithful distribution of genetic material, respectively. The ability to identify and exchange the strands of two homologous DNA molecules lies at the heart of HR and is mediated by RecA-family recombinases. Dmc1 is a meiosis-specific RecA homolog in eukaryotes, playing a predominant role in meiotic HR. However, Dmc1 cannot function without its two major auxiliary factor complexes, Swi5-Sfr1 and Hop2-Mnd1. Through biochemical reconstitutions, we demonstrate that Swi5-Sfr1 and Hop2-Mnd1 make unique contributions to stimulate Dmc1-driven strand exchange in a synergistic manner. Mechanistically, Swi5-Sfr1 promotes establishment of the Dmc1 nucleoprotein filament, whereas Hop2-Mnd1 defines a critical, rate-limiting step in initiating strand exchange. Following execution of this function, we propose that Swi5-Sfr1 then promotes strand exchange with Hop2-Mnd1. Thus, our findings elucidate distinct yet complementary roles of two auxiliary factors in Dmc1-driven strand exchange, providing mechanistic insights into some of the most critical steps in meiotic HR.