Vladimir Potapov

Replicative DNA synthesis is distributed among several DNA polymerases (DNAPs) in both Bacteria and Eukarya. The division of synthesis permits specialized activities of unique DNAPs to facilitate rapid replication in a continuous manner on the leading strand and discontinuous synthesis of the lagging strand with repetitive priming and maturation of Okazaki fragments. Many archaea encode two evolutionarily unrelated DNAPs, PolB and PolD, but how leading and lagging strand synthesis responsibilities are partitioned between each polymerase has yet to be resolved in vivo. In the model archaeon Thermococcus kodakarensis, Pol B is dispensable, demonstrating PolD can independently support replicative synthesis, but does not exclude the possibility that PolB normally participates in key aspects of DNA replication. Here, we combine phenotypic analyses of strains encoding variant DNAPs, high-throughput sequencing, and purified replication assays to establish that PolB actively facilitates lagging strand synthesis in vivo. Pol B outperforms PolD at strand displacement and Okazaki fragment maturation, demonstrating that specialized DNAPs share replicative synthesis duties in most Archaea. Our findings define a foundational division of labor between PolB and PolD in Archaea and establish the use of specialized replicative DNAP activities for strand-specific synthesis across all life.

Nucleotide- and sequence-specific ribonucleases are essential tools for dissecting RNA structure, modification, and composition, yet the enzymatic repertoire available for high-resolution RNA analysis remains limited. Here, we systematically screened 186 MazF homologs, including 164 from archaeal organisms, and identified 132 active endoribonucleases with diverse sequence preferences. Using a semi-high-throughput expression and purification workflow combined with an Illumina-based RNA-seq assay, we defined recognition motifs for 90 enzymes, uncovering a striking diversity of UG-rich cleavage specificities across the archaeal domain. Among these, Tth-MazF1, a thermostable endoribonuclease from the hyperthermophilic archaeon Thermococcus thioreducens, displayed robust and specific cleavage at U↓GN motifs across a broad range of temperatures (4°C–85°C). Tth-MazF1 enabled high sequence coverage of 0.8–8.9 kb messenger RNA (mRNA) transcripts by LC-MS/MS and direct detection of key structural features, including the 5′ cap, poly(A) tail, and mass-altering epitranscriptomic modifications in ribosomal RNA. Tolerance of Tth-MazF1 to high temperatures, salt, and chemical modifications highlights it as a powerful enzymatic tool for analyzing chemically modified and structured RNAs. These findings position archaeal MazF proteins as a rich source of customizable RNases for biotechnology applications, such as mass spectrometry-based analysis of RNA-based vaccines and therapeutics as well as cellular RNAs.

Bacteriophages have applications in biotechnology, including human and veterinary medicine, agriculture, food safety, and biosecurity. One example of resurging importance is phage therapy, the use of phages to treat antibiotic-resistant bacterial infections. Phage therapy currently requires screening of environmental phages against the infecting strains for each individual patient, a laborious process that limits the development of standardized treatments. To overcome limitations of narrow host range inherent to many native phages, a robust genomic engineering platform is required which permits rapid and dependable genome production and engineering for nonmodel phage. Here, we describe an engineering platform for a phiKMV-like Pseudomonas aeruginosa phage, 41S1 that builds on previous work in the rapid assembly of small genomes through one-pot, High Complexity Golden Gate assembly (HC-GGA). This system divides the 41S1 genome into DNA fragments small enough to be conveniently synthesized and to avoid toxicity during DNA propagation, with all but one maintained in Escherichia coli . These fragments are readily assembled in a high accuracy, one-pot reaction; phages can be rescued by direct transformation into P. aeruginosa PAO1 or E. coli 10-beta cells. We demonstrate the precise generation of point mutations, DNA insertions, deletions, and the addition of fluorescence reporter genes that are expressed during phage replication. All viable genotypes could be generated with near 100% success rate with minimal screening. This system demonstrates the potential of HC-GGA for the rapid production and engineering of phages and provides a chassis for the development of phages that broadly target the opportunistic human pathogen P. aeruginosa .

DNA polymerases (DNAPs) are indispensable enzymes that play central roles in biology, by replicating and repairing genetic material, as well as in biotechnology, by fueling such innovations as polymerase chain reaction (PCR), cloning, and DNA sequencing. Replicative DNAPs possess dual catalytic activities that work together for high accuracy replication: a selective DNA-dependent DNA polymerase activity for synthesizing DNA and a proofreading exonuclease activity for removing misincorporated nucleotides. Despite their precision, DNA polymerases occasionally make errors, and understanding the mechanisms behind these mistakes is essential to fully leverage these enzymes. Indeed, measuring DNA polymerase fidelity not only reveals the basis of their accuracy, but also enables rational modulation of their fidelity. Here we employ a highly accurate Pacific Biosciences sequencing workflow that leverages long-read, non-PCR-based technology to measure DNAP error rates and profiles. By measuring the fidelity of the four primary replicative DNA polymerase families, A, B, C, and D, measurements uncovered remarkably diverse family specific error profiles. Factors that influence DNAP fidelity, such as deoxynucleoside triphosphate ratios, replication components, and exonuclease and polymerase active site mutations, are further explored. This work deepens our understanding of DNA replication, the mechanisms that underly DNA polymerase fidelity, and informs development of advanced DNA polymerase-based tools for biotechnology.

Commercially synthesized genes are typically made using variations of homology-based cloning techniques, including polymerase cycling assembly from chemically synthesized microarray-derived oligonucleotides. Here we apply Data-optimized Assembly Design to the synthesis of hundreds of codon-optimized genes in both constitutive and inducible vectors using Golden Gate Assembly. Starting from oligonucleotide pools, we synthesize genes in three simple steps: 1) Amplification of parts belonging to individual assemblies in parallel from a single pool; 2) Golden Gate Assembly of parts for each construct; and 3) Transformation. We construct genes from receiving DNA to sequence confirmed isolates in as little as 4 days. By leveraging the ligation fidelity afforded by T4 DNA ligase, we expect to be able to construct a larger breadth of sequences not currently supported by homology-based methods which require stability of extensive single-stranded DNA overhangs.

DNA ligases, critical enzymes for in vivo genome maintenance and modern molecular biology, catalyze the joining of adjacent 3′-OH and 5′-phosphorylated ends in DNA. To determine whether DNA annealing equilibria or properties intrinsic to the DNA ligase enzyme impact end-joining ligation outcomes, we used a highly multiplexed, sequencing-based assay to profile mismatch discrimination and sequence bias for several ligases capable of efficient end-joining. Our data reveal a spectrum of fidelity and bias, influenced by both the strength of overhang annealing as well as sequence preferences and mismatch tolerances that vary both in degree and kind between ligases. For example, while T7 DNA ligase shows a strong preference for ligating high GC sequences, other ligases show little GC-dependent bias, with human DNA Ligase 3 showing almost none. Similarly, mismatch tolerance varies widely among ligases, and while all ligases tested were most permissive of G:T mismatches, some ligases also tolerated bulkier purine:purine mismatches. These comprehensive fidelity and bias profiles provide insight into the biology of end-joining reactions and highlight the importance of ligase choice in application design.

Prokaryotic Argonautes (pAgos) use small nucleic acids as specificity guides to cleave single-stranded DNA at complementary sequences. DNA targeting function of pAgos creates attractive opportunities for DNA manipulations that require programmable DNA cleavage. Currently, the use of mesophilic pAgos as programmable endonucleases is hampered by their limited action on double-stranded DNA (dsDNA). We demonstrate here that efficient cleavage of linear dsDNA by mesophilic Argonaute CbAgo from Clostridium butyricum can be activated in vitro via the DNA strand unwinding activity of nuclease deficient mutant of RecBC DNA helicase from Escherichia coli (referred to as RecB^exo–C). Properties of CbAgo and characteristics of simultaneous cleavage of DNA strands in concurrence with DNA strand unwinding by RecB^exo–C were thoroughly explored using 0.03–25 kb dsDNAs. When combined with RecB^exo–C, CbAgo could cleave targets located 11–12.5 kb from the ends of linear dsDNA at 37°C. Our study demonstrates that CbAgo with RecB^exo–C can be programmed to generate DNA fragments with custom-designed single-stranded overhangs suitable for ligation with compatible DNA fragments. The combination of CbAgo and RecB^exo–C represents the most efficient mesophilic DNA-guided DNA-cleaving programmable endonuclease for in vitro use in diagnostic and synthetic biology methods that require sequence-specific nicking/cleavage of linear dsDNA at any desired location.

Prokaryotic Argonautes (pAgos) use small nucleic acids as specificity guides to cleave single-stranded DNA at complementary sequences. DNA targeting function of pAgos creates attractive opportunities for DNA manipulations that require programmable DNA cleavage. Discovery of mesophilic Argonautes active at physiological temperature places pAgos closer to their possible application for genome editing as a simpler alternative to CRISPR/Cas nucleases. Currently, the use of mesophilic pAgos as programmable DNA endonucleases is hampered by their poor action on double-stranded DNA (dsDNA), mainly due to their inability to invade the DNA duplex. The present study demonstrates that efficient in vitro cleavage of double-stranded DNA by mesophilic Argonaute CbAgo from Clostridium butyricum can be activated via the DNA strand unwinding activity of nuclease deficient mutant of RecBC DNA helicase from Escherichia coli (referred to as RecB ^exo- C). Properties of CbAgo and characteristics of simultaneous cleavage of complementary DNA strands in concurrence with DNA strand unwinding by RecB ^exo- C were thoroughly explored using 0.3-25 kb DNA substrates. When combined with RecB ^exo- C helicase, CbAgo was capable of cleaving target sequences located 11-12.5 kb from the ends of linear dsDNA at 37ºC. Our study demonstrates that CbAgo with RecB ^exo- C can be programmed to generate dsDNA fragments flanked with custom-designed single-stranded overhangs suitable for ligation with compatible DNA fragments. At present, the combination of CbAgo and RecB ^exo- C represents the most efficient mesophilic DNA-guided DNA-cleaving programmable endonuclease for use in diagnostic and synthetic biology methods that require sequence-specific nicking/cleavage of dsDNA at any desired location.

Large DNA constructs (>10 kb), including small genomes and artificial chromosomes, are invaluable tools for genetic engineering and vaccine development. However, the manufacture of these constructs is laborious. To address this problem, we applied new design insights and modified protocols to Golden Gate assembly. While this methodology is routinely used to assemble 5-10 DNA parts in one-step, we found that optimized assembly permitted >50 DNA fragments to be faithfully assembled in a single reaction. We applied these insights to genome construction, carrying out rapid assembly of the 40 kb T7 bacteriophage genome from 52 parts and recovering infectious phage particles after cellular transformation. The new Golden Gate assembly protocols and design principles described here can be applied to rapidly engineer a wide variety of large and complex assembly targets.

Next-generation sequencing technology has enabled the detection of rare genetic or somatic mutations and contributed to our understanding of disease progression and evolution. However, many next-generation sequencing technologies first rely on DNA amplification, via the Polymerase Chain Reaction (PCR), as part of sample preparation workflows. Mistakes made during PCR appear in sequencing data and contribute to false mutations that can ultimately confound genetic analysis. In this report, a single-molecule sequencing assay was used to comprehensively catalog the different types of errors introduced during PCR, including polymerase misincorporation, structure-induced template-switching, PCR-mediated recombination and DNA damage. In addition to well-characterized polymerase base substitution errors, other sources of error were found to be equally prevalent. PCR-mediated recombination by Taq polymerase was observed at the single-molecule level, and surprisingly found to occur as frequently as polymerase base substitution errors, suggesting it may be an underappreciated source of error for multiplex amplification reactions. Inverted repeat structural elements in lacZ caused polymerase template-switching between the top and bottom strands during replication and the frequency of these events were measured for different polymerases. For very accurate polymerases, DNA damage introduced during temperature cycling, and not polymerase base substitution errors, appeared to be the major contributor toward mutations occurring in amplification products. In total, we analyzed PCR products at the single-molecule level and present here a more complete picture of the types of mistakes that occur during DNA amplification.