Defining the Sequence Requirements for the Positioning of Base J in DNA Using SMRT Sequencing

Overview

Journal Nucleic Acids Res

Publisher Oxford University Press

Specialty Biochemistry

Date 2015 Feb 10

PMID 25662217

Citations 17

Authors

Paul-Andre Genest

Loren Baugh

Alex Taipale

Wanqi Zhao

Sabrina Jan

Henri G A M van Luenen

Jonas Korlach

Tyson Clark

Khai Luong

Matthew Boitano

Steve Turner

Peter J Myler

Piet Borst

Affiliations

Soon will be listed here.

Abstract

Base J (β-D-glucosyl-hydroxymethyluracil) replaces 1% of T in the Leishmania genome and is only found in telomeric repeats (99%) and in regions where transcription starts and stops. This highly restricted distribution must be co-determined by the thymidine hydroxylases (JBP1 and JBP2) that catalyze the initial step in J synthesis. To determine the DNA sequences recognized by JBP1/2, we used SMRT sequencing of DNA segments inserted into plasmids grown in Leishmania tarentolae. We show that SMRT sequencing recognizes base J in DNA. Leishmania DNA segments that normally contain J also picked up J when present in the plasmid, whereas control sequences did not. Even a segment of only 10 telomeric (GGGTTA) repeats was modified in the plasmid. We show that J modification usually occurs at pairs of Ts on opposite DNA strands, separated by 12 nucleotides. Modifications occur near G-rich sequences capable of forming G-quadruplexes and JBP2 is needed, as it does not occur in JBP2-null cells. We propose a model whereby de novo J insertion is mediated by JBP2. JBP1 then binds to J and hydroxylates another T 13 bp downstream (but not upstream) on the complementary strand, allowing JBP1 to maintain existing J following DNA replication.

Citing Articles

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Obara P, Wolski P, Panczyk T Molecules. 2024; 29(19).

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Distant sequence regions of JBP1 contribute to J-DNA binding.

de Vries I, Ammerlaan D, Heidebrecht T, Celie P, Geerke D, Joosten R Life Sci Alliance. 2023; 6(9).

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G-Quadruplexes as Key Transcriptional Regulators in Neglected Trypanosomatid Parasites.

Monti L, Di Antonio M Chembiochem. 2023; 24(12):e202300265.

PMID: 37146230 PMC: 10946822. DOI: 10.1002/cbic.202300265.

Behind Base J: The Roles of JBP1 and JBP2 on Trypanosomatids.

Assis L, de Paiva S, Cano M Pathogens. 2023; 12(3).

PMID: 36986389 PMC: 10057400. DOI: 10.3390/pathogens12030467.

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