Capillary Electrophoresis-Based Functional Genomics Screening to Discover Novel Archaeal DNA Modifying Enzymes
| Autor: | Gregory J. S. Lohman, Léa Chuzel, Paul J Caffrey, Samantha L. Fossa, Andrew F. Gardner, Christopher H. Taron, Kelly M Zatopek, Katharina Bilotti, Alexandra M. Gehring |
|---|---|
| Rok vydání: | 2022 |
| Předmět: |
Ecology
biology Archaeal Proteins Electrophoresis Capillary Genomics Computational biology biology.organism_classification Applied Microbiology and Biotechnology DNA sequencing Thermococcus kodakarensis Thermococcus Fosmid genomic DNA chemistry.chemical_compound DNA Archaeal chemistry Escherichia coli Nucleic acid Genomic library Functional genomics DNA Food Science Biotechnology |
| Zdroj: | Applied and Environmental Microbiology. 88 |
| ISSN: | 1098-5336 0099-2240 |
| DOI: | 10.1128/aem.02137-21 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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