Capillary Electrophoresis-Based Functional Genomics Screening to Discover Novel Archaeal DNA Modifying Enzymes

Abstract

It has been predicted that 30-80% of archaeal genomes remain annotated as hypothetical proteins with no assigned gene function. Further, many archaeal organisms are difficult to grow or are unculturable. To overcome these technical and experimental hurdles, we have developed a high-throughput functional genomics screen that utilizes capillary electrophoresis (CE) to identify nucleic acid modifying enzymes based on activity rather than sequence homology. Here, we describe a functional genomics screening workflow to find DNA modifying enzyme activities encoded by the hyperthermophile Thermococcus kodakarensis ( T. kodakarensis ). Large DNA insert fosmid libraries representing a ∼5-fold average coverage of the T. kodakarensis genome were prepared in E. coli . RNA-seq showed a high fraction (84%) of T. kodakarensis genes were transcribed in E. coli despite differences in promoter structure and translational machinery. Our high-throughput screening workflow used fluorescently labeled DNA substrates directly in heat-treated lysates of fosmid clones with capillary electrophoresis detection of reaction products. Using this method, we identified both a new DNA endonuclease activity for a previously described RNA endonuclease (Nob1) and a novel AP lyase DNA repair enzyme family (termed 'TK0353') found only in a small subset of Thermococcales. The screening methodology described provides a fast and efficient way to explore the T. kodakarensis genome for a variety of nucleic acid modifying activities and may have implications for similar exploration of enzymes and pathways that underlie core cellular processes in other Archaea. IMPORTANCE This study provides a rapid, simple, high-throughput method to discover novel archaeal nucleic acid modifying enzymes by utilizing a fosmid genomic library, next-generation sequencing and capillary electrophoresis. The method described here provides details necessary to create 384-well fosmid library plates from Thermococcus kodakarensis genomic DNA, sequence 384-well fosmids plates using Illumina next generation sequencing and perform high-throughput functional read-out assays using capillary electrophoresis to identify a variety of nucleic acid modifying activities including DNA cleavage and ligation. We used this approach to identify a new DNA endonuclease activity for a previously described RNA endonuclease (Nob1) and identify a novel AP lyase enzyme (TK0353) that lacks sequence homology to known nucleic acid modifying enzymes.

High-throughput nanopore DNA sequencing of large insert fosmid clones directly from bacterial colonies

“…To illustrate the robustness of isothermal fosmid amplification, fosmids from a Thermococcus kodakarensis (T. kodakarensis) genomic library were amplified using phi29-XT DNA polymerase. The T. kodakarensis fosmid library was created previously by cloning ∼40 kb fragments of T. kodakarensis genomic DNA into pCC1FOS vector and using them to transform Escherichia coli EPI300 cells (19). Individual clones were then arrayed in a 384-well format.…”

“…Individual clones were then arrayed in a 384-well format. This fosmid library was formerly used in various functional screening assays and was originally sequenced and assembled from Illumina sequencing of libraries constructed using the plexWell TM 384 Library Preparation kit (seqWell, Danvers, MA) with fosmid DNA purified from liquid cultures as the input material (19). These sequences serve as a reference to demonstrate the efficacy of our workflow.…”

High-throughput Kinetics using Capillary Electrophoresis and Robotics (HiKER) platform used to Study T7, T3, and Sp6 RNA Polymerase Misincorporation

“…This methodology has shown potential in a variety of applications [61, 70-79]. The 3730 DNA Analyzer from ThermoFisher Scientific (Waltham, MA) was used in this study and processes 96 samples simultaneously.…”

“…CE is an opticsbased system where a dye must be present on the RNA/DNA for detection. This methodology has shown potential in a variety of applications [61,[70][71][72][73][74][75][76][77][78][79]. The 3730 DNA Analyzer from ThermoFisher Scientific (Waltham, MA) was used in this study and processes 96 samples simultaneously.…”

Thermococcus kodakarensis TK0353 is a novel AP lyase with a new fold

“…As a means to discover unannotated hyperthermophilic DNA repair enzymes, we recently reported ( 16 ) a high-throughput pipeline that utilizes a functional screen, as opposed to bioinformatic prediction, to uncover new gene function. This pipeline enabled the discovery of a novel AP lyase from the hyperthermophilic archaeon Thermococcus kodakarensis (Tko).…”