TET/JBP (ten-eleven translocation/base J binding protein) enzymes are iron(II)- and 2-oxo-glutarate–dependent dioxygenases that are found in all kingdoms of life and oxidize 5-methylpyrimidines on the polynucleotide level. Despite their prevalence, few examples have been biochemically characterized. Among those studied are the metazoan TET enzymes that oxidize 5-methylcytosine in DNA to hydroxy, formyl, and carboxy forms and the euglenozoa JBP dioxygenases that oxidize thymine in the first step of base J biosynthesis. Both enzymes have roles in epigenetic regulation. It has been hypothesized that all TET/JBPs have their ancestral origins in bacteriophages, but only eukaryotic orthologs have been described. Here we demonstrate the 5mC-dioxygenase activity of several phage TETs encoded within viral metagenomes. The clustering of these TETs in a phylogenetic tree correlates with the sequence specificity of their genomically cooccurring cytosine C5-methyltransferases, which install the methyl groups upon which TETs operate. The phage TETs favor Gp5mC dinucleotides over the 5mCpG sites targeted by the eukaryotic TETs and are found within gene clusters specifying complex cytosine modifications that may be important for DNA packaging and evasion of host restriction.

The study of complex immunological diseases and tumor microenvironments has advanced through recent developments in sequencing of the immune repertoire. Using this approach, the interrogation of disease progression is facilitated through analysis of millions of V(D)J combinations from B cell receptors (BCRs) and T cell receptors (TCRs). One major challenge of immune repertoire sequencing is to accurately capture the structural and sequence complexities of antibodies and TCR genes during both library preparation and bioinformatic analysis. In addition, the lack of standard reference controls makes assessing sequencing accuracy and sensitivity challenging. Here, we present a method for accurate sequencing of full-length immune gene repertoires of B cells and T cells. RNA extracted from peripheral blood mononuclear cells (PBMCs) or tissues were used for reverse transcription, during which unique molecular identifiers (UMIs) were added to discretely barcode each mRNA molecule. BCR- and TCR-specific PCR primers were used to enrich full-length BCR and TCR sequences. We have implemented a data analysis pipeline to assemble the full length BCR/TCR transcripts and to collapse PCR copies of each mRNA fragment into a single consensus sequence using UMIs. UMI incorporation enables the absolute quantification of input RNA molecules and accurate ranking of antibody/TCR clone abundance. Furthermore, we developed an RNA control pool that contains a large dynamic range of BCR and TCR sequences with a variety of V(D)J combinations, which enables sensitivity and accuracy evaluation of BCR and TCR sequencing. Our immune repertoire sequencing approach allows accurate clonal determination for both BCR and TCR. This technique is applicable for various applications including design of antibody chains for in vitro synthesis, investigation of T cell infiltration of tumor microenvironments, and monitoring of minimal residual disease in cancer patients. Citation Format: Chen Song, Ariel Erijman, Sean Lund, Bradley W. Langhorst, Pingfang Liu. Immune repertoire sequencing enables accurate clonality determination [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3304.

Bacteriophage enzymes synthesize varied and complex DNA hypermodifications. The enzyme encoded by the phage Mu genemomis necessary for post-replicative carbamoylmethyl addition to the exocyclic amine of deoxyadenosine in DNA during the lytic phase of the viral life-cycle. The molecular details of this modification reaction, including the molecular origins of the modification itself, have long eluded understanding. Here, we demonstrate that Mom co-opts the translational machinery of the host by harvesting activated glycine from charged tRNAGlyto hypermodify adenine. Based on this insight, we report the firstin vitroreconstitution of the Mu hypermodification from purified components. Using isotope labeling, we demonstrate that the carbamoyl nitrogen of the Mom modification is derived from theN6 of adenine, indicating an on-base rearrangement of theN6 aminoacylation product, possibly via a cyclic intermediate. Informed by the X-ray crystal structure of Mom, we have probed the location of the active site, identified a novel insertion, and established substrate specificities of the Mom enzyme.