Defining the sequence requirements for the positioning of base J in DNA using SMRT sequencing

Genest, Paul-André; Baugh, Loren; Taipale, Alex; Zhao, Weihua; Jan, Sabrina; Luenen, Henri G.A.M. van; Korlach, Jonas; Clark, Tyson A.; Luong, Khai; Boitano, Matthew; Turner, Steve; Myler, Peter J.; Borst, Piet

doi:10.1093/nar/gkv095

Cited by 27 publications

(30 citation statements)

References 49 publications

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“…within an episome instead of within the genome, DNA sequence specificity of J synthesis is maintained. This was also seen using a similar approach in L. tarentolae [17]. …”

supporting

confidence: 62%

“…It is unclear what determines the specific localization of base J synthesis into specific sequences in the genome. While no consensus sequence or motif is evident from the genome-wide J analysis thus far, it is clear that, for at least the telomeric repeats, there is a sequence specificity component where in the top strand (GGGTTA), only the second T is modified [17, 18]. …”

mentioning

confidence: 99%

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Base J glucosyltransferase does not regulate the sequence specificity of J synthesis in trypanosomatid telomeric DNA

Bullard

Cliffe

Wang³

et al. 2015

Molecular and Biochemical Parasitology

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Telomeric DNA of Trypanosomatids possesses a modified thymine base, called base J, that is synthesized in a two-step process; the base is hydroxylated by a thymidine hydroxylase forming hydroxymethyluracil (hmU) and a glucose moiety is then attached by the J-associated glucosyltransferase (JGT). To examine the importance of JGT in modifiying specific thymine in DNA, we used a Leishmania episome system to demonstrate that the telomeric repeat (GGGTTA) stimulates J synthesis in vivo while mutant telomeric sequences (GGGTTT, GGGATT, and GGGAAA) do not. Utilizing an in vitro GT assay we find that JGT can glycosylate hmU within any sequence with no significant change in Km or kcat, even mutant telomeric sequences that are unable to be J-modified in vivo. The data suggests that JGT possesses no DNA sequence specificity in vitro, lending support to the hypothesis that the specificity of base J synthesis is not at the level of the JGT reaction.

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“…within an episome instead of within the genome, DNA sequence specificity of J synthesis is maintained. This was also seen using a similar approach in L. tarentolae [17]. …”

supporting

confidence: 62%

mentioning

confidence: 99%

Base J glucosyltransferase does not regulate the sequence specificity of J synthesis in trypanosomatid telomeric DNA

Bullard

Cliffe

Wang³

et al. 2015

Molecular and Biochemical Parasitology

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“…For any single-molecule, kinetic-based method (ONT, PacBio, Genia), there is an ability to detect epigenetic states, including methyl-cytosine, methyl-6adenosize, and hydroxyl-methyl cytosine, with a potential for detecting a variety of other nucleic acid variants, such as those from DNA damage (e.g., 8-oxo-guanosine). [55][56][57][58][59] Synthetically phased library preparations…”

Section: Natively Phased Library Preparation Methodsmentioning

confidence: 99%

International Standards for Genomes, Transcriptomes, and Metagenomes

et al. 2017

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Challenges and biases in preparing, characterizing, and sequencing DNA and RNA can have significant impacts on research in genomics across all kingdoms of life, including experiments in single-cells, RNA profiling, and metagenomics (across multiple genomes). Technical artifacts and contamination can arise at each point of sample manipulation, extraction, sequencing, and analysis. Thus, the measurement and benchmarking of these potential sources of error are of paramount importance as next-generation sequencing (NGS) projects become more global and ubiquitous. Fortunately, a variety of methods, standards, and technologies have recently emerged that improve measurements in genomics and sequencing, from the initial input material to the computational pipelines that process and annotate the data. Here we review current standards and their applications in genomics, including whole genomes, transcriptomes, mixed genomic samples (metagenomes), and the modified bases within each (epigenomes and epitranscriptomes). These standards, tools, and metrics are critical for quantifying the accuracy of NGS methods, which will be essential for robust approaches in clinical genomics and precision medicine.

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“…From what we know about the mechanism of J biosynthesis, it follows that the highly restricted distribution of J-base must be co-determined by the thymidine hydroxylases (JBP1 and JBP2) that catalyze the initial step in J synthesis. Using SMRT sequencing of DNA segments inserted into plasmids grown in Leishmania 19 , it has been shown that J modification usually occurs near G-rich sequences potentially capable of forming G-quadruplexes and at pairs of Ts on opposite DNA strands, separated by 12 nucleotides. That led Genest et al 19 to propose a model in which JBP2 is responsible for initial J synthesis; then JBP1 binds to pre-existing J and hydroxylates another T that typically resides 13 bp downstream (but not upstream) on the complementary DNA strand.…”

Section: Introductionmentioning

confidence: 99%

“…Using SMRT sequencing of DNA segments inserted into plasmids grown in Leishmania 19 , it has been shown that J modification usually occurs near G-rich sequences potentially capable of forming G-quadruplexes and at pairs of Ts on opposite DNA strands, separated by 12 nucleotides. That led Genest et al 19 to propose a model in which JBP2 is responsible for initial J synthesis; then JBP1 binds to pre-existing J and hydroxylates another T that typically resides 13 bp downstream (but not upstream) on the complementary DNA strand. This model provides a mechanism explaining how JBP1 can maintain existing J following DNA replication.…”

Section: Introductionmentioning

confidence: 99%

The domain architecture of JBP1 suggests synergy between J-base DNA binding and thymidine hydroxylase activity

Adamopoulos

Heidebrecht

Roosendaal

et al. 2018

Preprint

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JBP1 (J-DNA Binding Protein 1) contributes to biosynthesis and maintenance of base J (β-D-glucosylhydroxymethyluracil), a modification of thymidine (T) confined to pathogenic protozoa. JBP1 has two known functional domains: an N-terminal thymidine hydroxylase (TH) homologous to the 5methylcytosine hydroxylase domain in TET proteins; and a J-DNA binding domain (JDBD) that resides in the middle of JBP1. Here we show that removing JDBD from JBP1 results in a soluble protein (Δ-JDBD) with the N-and C-terminal regions tightly associated together in a well-ordered domain. This Δ-JDBD domain retains thymidine hydroxylation activity in vitro, but displays a fifteen-fold lower apparent rate of hydroxylation compared to JBP1. Small Angle X-ray Scattering (SAXS) experiments on JBP1 and JDBD in the presence and absence of J-DNA, and on Δ-JDBD, allowed us to generate low-resolution three-dimensional models. We conclude that Δ-JDBD, and not the N-terminal region of JBP1 alone, is a distinct folding unit. Our SAXS-based model supports the notion that binding of JDBD specifically to J-DNA can facilitate hydroxylation a T 12-14 bp downstream on the complementary strand of the J-recognition site. We postulate that insertion of the JDBD module in the Δ-JDBD scaffold during evolution provided a mechanism to synergize between J recognition and T hydroxylation, ensuring inheritance of J in specific sequence patterns following DNA replication.

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Defining the sequence requirements for the positioning of base J in DNA using SMRT sequencing

Cited by 27 publications

References 49 publications

Base J glucosyltransferase does not regulate the sequence specificity of J synthesis in trypanosomatid telomeric DNA

Base J glucosyltransferase does not regulate the sequence specificity of J synthesis in trypanosomatid telomeric DNA

International Standards for Genomes, Transcriptomes, and Metagenomes

The domain architecture of JBP1 suggests synergy between J-base DNA binding and thymidine hydroxylase activity

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