Thermococcus kodakarensis TK0353 is a novel AP lyase with a new fold
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Biochemistry & Molecular Biology↗
Engineering topics
Burkhart, Brett W.
Publications and source records attributed to Burkhart, Brett W..
The Hyperthermophilic Restriction-Modification Systems of Thermococcus kodakarensis Protect Genome Integrity
Thermococcus kodakarensis (T. kodakarensis), a hyperthermophilic, genetically accessible model archaeon, encodes two putative restriction modification (R-M) defense systems, TkoI and TkoII. TkoI is encoded by TK1460 while TkoII is encoded by TK1158. Bioinformative analysis suggests both R-M enzymes are large, fused methyltransferase (MTase)-endonuclease polypeptides that contain both restriction endonuclease (REase) activity to degrade foreign invading DNA and MTase activity to methylate host genomic DNA at specific recognition sites. In this work, we demonsrate T. kodakarensis strains deleted for either or both R-M enzymes grow more slowly but display significantly increased competency compared to strains with intact R-M systems, suggesting that both TkoI and TkoII assist in maintenance of genomic integrity in vivo and likely protect against viral- or plasmid-based DNA transfers. Pacific Biosciences single molecule real-time (SMRT) sequencing of T. kodakarensis strains containing both, one or neither R-M systems permitted assignment of the recognition sites for TkoI and TkoII and demonstrated that both R-M enzymes are TypeIIL; TkoI and TkoII methylate the N 6 position of adenine on one strand of the recognition sequences GTGAAG and TTCAAG, respectively. Further in vitro biochemical characterization of the REase activities reveal TkoI and TkoII cleave the DNA backbone GTGAAG(N) 20 /(N) 18 and TTCAAG(N) 10 /(N) 8 , respectively, away from the recognition sequences, while in vitro characterization of the MTase activities reveal transfer of tritiated S-adenosyl methionine by TkoI and TkoII to their respective recognition sites. Together these results demonstrate TkoI and TkoII restriction systems are important for protecting T. kodakarensis genome integrity from invading foreign DNA.
59 BASIC BIOLOGICAL SCIENCES↗
Dynamic RNA acetylation revealed by quantitative cross-evolutionary mapping
N 4 -acetylcytidine (ac 4 C) is an ancient and highly conserved RNA modification that is present on tRNA and rRNA and has recently been investigated in eukaryotic mRNA. However, the distribution, dynamics and functions of cytidine acetylation have yet to be fully elucidated. In this work, we report ac 4 C-seq, a chemical genomic method for the transcriptome-wide quantitative mapping of ac 4 C at single-nucleotide resolution. In human and yeast mRNAs, ac 4 C sites are not detected but can be induced—at a conserved sequence motif—via the ectopic overexpression of eukaryotic acetyltransferase complexes. By contrast, cross-evolutionary profiling revealed unprecedented levels of ac 4 C across hundreds of residues in rRNA, tRNA, non-coding RNA and mRNA from hyperthermophilic archaea. Ac 4 C is markedly induced in response to increases in temperature, and acetyltransferase-deficient archaeal strains exhibit temperature-dependent growth defects. Visualization of wild-type and acetyltransferase-deficient archaeal ribosomes by cryo-electron microscopy provided structural insights into the temperature-dependent distribution of ac 4 C and its potential thermoadaptive role. Our studies quantitatively define the ac 4 C landscape, providing a technical and conceptual foundation for elucidating the role of this modification in biology and disease.
59 BASIC BIOLOGICAL SCIENCES↗
Archaeosine Modification of Archaeal tRNA: Role in Structural Stabilization
Archaeosine is ubiquitous in archaeal tRNA, where it is located at position 15. Based on its molecular structure, it was proposed to stabilize tRNA, and we show that loss of archaeosine in Thermococcus kodakarensis results in a strong temperature-sensitive phenotype, while there is no detectable phenotype when it is lost in Methanosarcina mazei . Measurements of tRNA stability show that archaeosine stabilizes the tRNA structure but that this effect is much greater when it is present in otherwise unmodified tRNA transcripts than in the context of fully modified tRNA, suggesting that it may be especially important during the early stages of tRNA processing and maturation in thermophiles. Our results demonstrate how small changes in the stability of structural RNAs can be manifested in significant biological-fitness changes.
Microbiology↗
Biochemical reconstitution and genetic characterization of the major oxidative damage base excision DNA repair pathway in Thermococcus kodakarensis
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59 BASIC BIOLOGICAL SCIENCES↗